Modified pigment epithelium-derived factor (PEDF) peptides and uses thereof for treating neovascular diseases, inflammatory diseases, cancer, and for cytoprotection

ABSTRACT

Disclosed are modified pigment epithelium-derived factor (PEDF) peptides, particulate carrier prodrugs thereof, and pharmaceutical compositions comprising the peptides or particulate carrier prodrugs. The peptides, particulate carrier prodrugs, and pharmaceutical compositions may be used to treat diseases and disorders that are amenable to treatment with anti-angiogenic agents, anti-tumorigenic agents, anti-fibrotic agents, chemotherapy-protecting agents, and immune stimulating agents.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application claims the benefit of priority under 35 U.S.C. 119(e)to U.S. Provisional Application No. 62/327,767, filed on Apr. 26, 2016,the content of which is incorporated herein by reference in itsentirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under R24 EY022883(University of Wisconsin Subcontract to Northwestern University,#457K273) awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND

The field of the invention relates to therapeutic peptides and prodrugsthereof. In particular, the field of the invention relates totherapeutic peptides which duplicate some of the beneficial effects ofpigment epithelium-derived factor (PEDF), to which they are related,including anti-angiogenic, anti-tumorigenic, and immunomodulatoryproperties.

Pigment epithelium-derived factor (PEDF) also known as serpin F1(SERPINF1), is a multifunctional secreted protein that hasanti-angiogenic, anti-tumorigenic, immunomodulatory and neurotrophicfunctions. PEDF is being researched as a therapeutic candidate fortreatment of such conditions as choroidal neovascularization, heartdisease, and cancer. Previously, an 18 amino-acid PEDF peptide, P18, wasshown to slow cancer growth and blocks ocular angiogenesis. We nowreport, more potent, practical and safe, modified peptides related toP18, which inhibit angiogenesis, directly kill ovarian cancer cells, andosteosarcoma cell, and exhibit other therapeutic properties. Themodified peptides are uniquely suited chemically to be formulated asprodrugs where the latter are attached to particulate carriers, such asnanoparticles, via labile bonds which can be selected to control therate of release of the modified peptides from the particulate carrier.The modified peptides also have amino acid substitutions that improvepotency as compared with the naturally occurring PEDF amino acidsequence. In addition, we now show that these peptides also enhanceresistance of the immune system to cancers, directly kill ovarian cancercells and bone cancer cells, and protect sensitive normal cells, such askidney cells, or retinal epithelium cells, from toxic effects ofchemotherapy. The peptides also are shown to mitigate fibrotic processesand to have immune stimulatory activity.

SUMMARY

Disclosed are modified pigment epithelium-derived factor (PEDF)peptides, particulate carrier prodrugs thereof, and pharmaceuticalcompositions comprising the peptides or particulate carrier prodrugs.The peptides, particulate carrier prodrugs, and pharmaceuticalcompositions may be used to treat diseases and disorders that areamenable to treatment with anti-angiogenic agents, anti-tumorigenicagents, anti-fibrotic agents, and immune stimulating agents.

The disclosed peptides are modified peptides comprising an N-terminalcarboxyl group. The modified peptides disclosed herein may be describedas having a modified amino acid sequence as follows:Z-B-X-AA0-AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9-AA10-Y  (SEQ ID NO:14)

where:

AA0, AA1, AA2, AA3, AA4, AA5, AA6, AA7, AA8, and AA9 are selected from anaturally occurring amino acid, including sarcosine, and beta alanine;

AA0 is absent or present, and when AA0 is present AA0 is selected fromsarcosyl and Gly; or beta alanine or beta alaninyl;

AA1 is absent or present, and when AA1 is present AA1 is selected fromsarcosyl and Gly;

AA2 is absent or present, and when AA2 is present AA2 optionally is Tyr;

AA3 is Asp or Asn, optionally when AA2 is present;

AA4 is Leu or Val;

AA5 is Tyr or Phe;

AA6 is Arg;

AA7 is Val or Pro;

AA8 is present or absent, and when AA8 is present AA8 is selected fromArg, Pro, and Gln;

AA9 is present or absent, and when AA9 is present AA9 selected from Serand Ala;

AA10 is absent or present and selected from Ser, Ser-Thr, Ser-Thr-Ser,Ser-Thr-Ser-Pro, and Ser-Thr-Ser-Pro-Thr;

X is absent or present, and when X is present X is selected from thegroup consisting of acetyl, butyryl, hexanoyl, methoxy-PEG_((n))CO,hydroxy-PEG_((n))CO, amino-PEG_((n))CO, and sarcosyl-amino-PEG_((n))CO,where n is 3-13, and X is in an amide bond to an amino terminus of AA0,AA1, or AA2;

B is a di-carboxylic acid containing from 4-8 carbon atoms, which may bestraight-chain or branched, and B is in a half-amide bond to an aminoterminus of X, or B is in a half-amide bond to an amino terminus of AA0when X is absent, or B is in a half-amide bond to an amino terminus ofAA1 when X and AA0 are absent, or B is in a half-amide bond to an aminoterminus of AA2 when X, AA0 and AA1 are absent, or B is in a half-amidebond to an amino terminus of AA3 when X, AA0, AA1, and AA2 are absent,and B may end in a free carboxyl group, or this otherwise free carboxylgroup may be in an ester bond to a hydroxyl group of Z;

Z is absent or present, and when Z is present Z is selected from thegroup consisting of: primary or secondary hydroxyl groups of sugarmonomers present on a polymeric carbohydrate carrier; hydroxyl groups ofa hydroxyl terminal dendrimer; and primary or secondary hydroxyl groupsof acyclic or cyclic amino alcohols having between 4 and 6 carbon atomsand a single primary or secondary amine that is protected through anamide bond or a carbamate bond; and Y is an amide or a substituted amideselected from alkylamide, dialkylamide, and PEG_((n))-amide, where n is4-12.

Also disclosed herein are prodrugs of the foregoing modified peptides inwhich the free carboxyl group of the disclosed peptides (e.g., a peptidehaving a formula B-X-AA0-AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9-AA10-Y (SEQID NO:14)) is esterified to a free hydroxyl group of a particulatecarrier (e.g., a nanoparticle carrier) to form a particulate carrierprodrug of the modified peptides. Optionally, the prodrugs comprise alinker (or a bridge structure, e.g., a peptide having a formulaZ-B-X-AA0-AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9-AA10-Y (SEQ ID NO:14)) forlinking the peptide to a nanoparticle carrier. The linker may comprise afree hydroxyl group and a free amino group (e.g., an amino alcohollinker). As such, the foregoing modified peptides may be esterified tothe linker via the free hydroxyl group and the linker may be attached toa nanoparticle carrier via the amino group of the linker and a freehydroxyl group of the nanoparticle carrier forming a carbamate bond orvia the free amino group of the linker and a free carboxyl group of thenanoparticle carrier forming an amide bond.

Also disclosed herein are pharmaceutical compositions comprising any ofthe foregoing modified peptides or particulate carrier prodrugs thereof.The pharmaceutical compositions may be administered to a subject in atreatment method for treating diseases or disorders that are treated byanti-angiogenic agents and/or anti-tumorigenic agents, including but notlimited cell proliferative disorders such as cancer, eye disorders anddiseases, kidney diseases and disorders, ear diseases and disorders,inflammatory diseases and disorders, diseases or disorders that areexacerbated by immune suppression or fibrosis.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Novel modified peptides and new peptide sequences as exemplifiedby SEQ ID NOs:1-13. The ED50 (50% effective dose), is the measure ofanti-angiogenic potency obtained by quantitative assessment of apoptosisof activated human endothelial cells (EC) as described in FIGS. 3 and 4.

FIG. 2. Selective killing of the ovarian cancer cells by SEQ ID NO:2.Ovarian cancer cells (ID-8, OVCAR-3 and SKOV-3 cell lines), normalovarian epithelial cells (nOSE) and unstimulated mouse endothelial cells(mEC) were treated with increasing concentrations of SEQ ID NO:2 andsurvival was measured using a WST assay.

FIG. 3. Relative potency of peptides at inducing endothelial cell (EC)apoptosis. Human microvascular EC (HMVEC) were seeded on glasscoverslips and grown in EGM, supplemented with defined growth factorsand full serum (5%) to 80-90% confluence. The cells were transferredovernight in the medium with 1% serum, w/o growth factors. VEGF (10ng/ml) was added to induce pro-angiogenic function/survival. Peptideswere added at indicated concentrations. After 48 h the cells were fixedin 1% buffered paraformaldehyde and apoptosis detected by in situ TUNEL.Apoptotic cells were quantified in ˜10 10× fields per condition andapoptotic fraction (apoptotic/total cells) calculated for eachcondition. Apoptosis induced in endothelial cells by indicated peptides(SEQ ID NO:6, 8, 10, and 11) is compared to SEQ ID NO:4.

FIG. 4. Selective cytotoxic activity of SEQ. ID NO:4 in proliferatingendothelium and cancer cells. EC apoptosis was measured as in FIG. 3.Panel (A) Representative images of ECs treated with SEQ ID NO:4. VEGF ispresent in all conditions and apoptosis is assessed by TUNEL(fluorescence) and all cells are labeled with DAPI (nuclear marker).Panel (B) Specific pro-apoptotic activity of SEQ ID NO:4 inVEGF-stimulated endothelial cells but not in quiescent endothelium (lowserum). Panel (C) Specific proapoptitc activity in ovarian cancer cellsin serum-free media with and without SEQ ID NO:4. Cells in full serum(10%) are shown for comparison. Panel (D) Specific proapoptitc activityin ovarian cancer cells (OvCar3) but not in normal ovarian epithelialcells (NOECs) induced by SEQ ID NO:4. Ovarian cancer cells and normalovarian epithelial cells were grown to 70% confluence, transferred inserum-free medium and treated with indicated peptide concentrations for48 hrs.

FIG. 5. PEDF peptides inhibit the growth of orthotopic ovarian tumors.Mice were implanted with 10⁶ ovarian cancer cells in the left ovary andtumors allowed to reach progressive growth phase. The mice were treatedwith continuous peptide administration (5 mg/kg) for 4 weeks. At the endof treatment, mice were euthanized, the tumors extracted and processedfor analyses. Tumor growth quantitation was accomplished in real time bythe cells expressing a bioluminescent probe. Panel (A) Average tumorsweights at the time of sacrifice. Panel (A) shows the greater efficacyin vivo of SEQ ID NO:4 compared to SEQ ID NO:2, consistent with their invitro potencies. Panel (B) Representative tumor images form controlgroup and the group treated with SEQ ID NO:4. Panel B shows arepresentative difference in tumor size at endpoint. Panel (C)Representative histology in control and treated groups. Panel (C) showsnormal ovarian morphology in the ovaries of mice treated with SEQ IDNO:4. Panel (D, E) SEQ ID NO:4 blocks tumor angiogenesis and cancer celldivision in vivo. Ovarian tumors from mice treated with SEQ ID NO:4 orcontrol vehicle were stained for the marker of blood vessels (CD31) orproliferation marker (Ki-67). Note visible decreases in the number oftumor blood vessels (Panel (D)) and cell proliferation (Panel (E)) inthe presence of SEQ ID NO:4, compared to vehicle control.

FIG. 6. SEQ ID NO:4 enables mouse macrophages to kill tumor cells andreduces their immunosuppressive activity. Panels (A, B). Normal ovarianepithelial cells (NOE) and ovarian cancer cells (Ovcar-3) were culturedalone or admixed with macrophages (Raw264.7) at 1:3 ratio. All cellswere treated with SEQ ID NO:4 (10 nM) and cell death visualized (Panel(A)) and quantified (Panel B)) using TUNEL stain (green). Panel (C)Tumor cell killing was due to a macrophage-derived death molecule calledTRAIL (expression induced by SEQ ID NO:4). This was demonstrated byadding TRAIL inhibitor (decoy receptor-1 (DcR-1)) to the co-culture ofovarian cancer cells and macrophages and measuring cell death(Apoptosis) as in Panel (A). Panel (D) illustrates that SEQ ID NO:4attenuates immunosuppressive properties of tumor-associated macrophages.In ovarian cancer, macrophages are a rich source of immunosuppressivemolecule PD-L1, which interferes with ovarian cancer cells recognitionby T lymphocytes. Note that SEQ ID NO:4 reduces the expression of PD-L1by macrophages. Cell nuclei are highlighted.

FIG. 7. SEQ ID NO:4 promotes tumor-suppressive macrophages in vivo. Thisis illustrated in Panel (A) by decreased expression of mRNA for themarker of tumor-promoting macrophages (IL-10) and in Panel (B) byincreased expression of an mRNA encoding the marker for thetumor-suppressive phenotype (IL-12) in the macrophages cultured in thepresence of SEQ ID NO:4. Panel (C). Treatment with SEQ ID NO:4eliminates the tumor-promoting macrophages (top), while tumorsuppressive macrophages are prominent (bottom). Panel (D) illustratedthat treatment with SEQ ID NO:4 also increases macrophage infiltrationinto tumors. Panel (E) illustrates that, in tumors treated with SEQ IDNO:4, macrophages acquire the ability to kill tumor cells in theirproximity.

FIG. 8. PEDF peptides decrease viability of osteosarcoma cells. D17canine osteosarcoma cells were cultured under normal growth conditionsand treated with the indicated peptides/concentrations for 24 hours.Cell viability was measured by WST-1 assay. Consistent with theirrelative potency in other cell killing assays (see above), SEQ. ID NO:10and Seq. ID NO:11 show higher potency than SEQ. ID NO:4 and SEQ. IDNO:8. This also shows that both canine cancer cells and human cancercells are killed by modified PEDF peptides in FIG. 1.

FIG. 9. Peptide inhibition of laser-induced choroidal neovascularization(CNV). Mice were anesthetized for eye injections and were injectedintravitreally with 1 μl PBS vehicle control or with 1 μl of 4 mg/mlpeptide solution. After 2 days the same mice were re-anesthetized and 3local retinal laser burns introduced in each eye to induce choroidalneovascularization (CNV). At 14 days post induction, CNV was measured onchoroidal-scleral flat mounts using fluorescent antibody stain forICAM2. PECAM-1 positive CNV area was digitally measured to quantifytreatment responses. Panel (A), control, SEQ ID NO:8 and SEQ ID NO:4.Panel (B), control, SEQ ID NO:10 and SEQ ID NO:11.

FIG. 10. SEQ ID NO:10 mitigates laser-induced CNV, when administeredeither by intra-ocular injection, or as eye drops. SEQ. ID NO:10 (4mg/ml) or saline were injected 3 days prior to CNV induction. In aparallel group, mice were treated twice daily with eye drops containingSEQ. ID NO:10 (10 mg/ml) 7 days prior to CNV induction. After laserinduction, eye were continued for 11 days, with final analysis at day 14post induction. Both delivery methods significantly decreased CNV.

FIG. 11. PEDF peptides protect retinal and renal cells fromenvironmental insults. Panel (A) Protection of retinal pigmentepithelial cells (RPE-1) against peroxide-induced toxicity. Panel (B)Protective effects in renal cells (podocytes) from toxic effects ofhyperglycemic stress. High glucose (20 mM) and normal (5 mM) glucoseconditions are shown for comparison.

FIG. 12. SEQ ID NO:4 inhibits production of toxic reactive oxygenspecies (ROS). ROS were visualized using green fluorescent stain. Toprow: ROS production in renal cells (podocytes) from in normal (NG, 5 mM)and high glucose (20 mM, HG) conditions are shown for comparison and SEQID NO:4 added to high glucose condition. Bottom row: ROS production bypodocytes treated with chemotherapy agent, cisplatin (CPT) in thepresence and absence of SEQ ID NO:4 peptide.

FIG. 13. SEQ ID NO:4 inhibits fibroblast activation. Human primarydermal fibroblasts were activated with TGF-β1 (10 ng/ml), SEQ ID NO:4(30 nM) was added where indicated and PEDF (20 nM) was used as control.After 24 h the cells were lysed and activation assessed asphosphorylation of pSMAD. Note ˜5-fold decrease in pSMAD by SEQ ID NO:4.

FIG. 14. Amino alcohol ester-bridging groups.

FIG. 15. Activation of dextran-based carrier for direct ester formationof carboxypeptides.

DETAILED DESCRIPTION

The present invention is described herein using several definitions, asset forth below and throughout the application.

Unless otherwise specified or indicated by context, the terms “a”, “an”,and “the” mean “one or more.” For example, a “peptide” should beinterpreted to mean “one or more peptides.”

As used herein, “about,” “approximately,” “substantially,” and“significantly” will be understood by persons of ordinary skill in theart and will vary to some extent on the context in which they are used.If there are uses of these terms which are not clear to persons ofordinary skill in the art given the context in which they are used,“about” and “approximately” will mean plus or minus ≤10% of theparticular term and “substantially” and “significantly” will mean plusor minus >10% of the particular term.

As used herein, the terms “include” and “including” have the samemeaning as the terms “comprise” and “comprising.” The terms “comprise”and “comprising” should be interpreted as being “open” transitionalterms that permit the inclusion of additional components further tothose components recited in the claims. The terms “consist” and“consisting of” should be interpreted as being “closed” transitionalterms that do not permit the inclusion of additional components otherthan the components recited in the claims. The term “consistingessentially of” should be interpreted to be partially closed andallowing the inclusion only of additional components that do notfundamentally alter the nature of the claimed subject matter.

As used herein, a “subject” may be interchangeable with a “patient” or“individual” and means an animal, which may be a human or non-humananimal, in need of treatment. Non-human animals may include dogs, cats,horses, cows, pigs, sheep, and the like.

A “subject in need thereof” may include a subject having or at risk fordeveloping a cell proliferative disorder such as cancer. Individuals whoare treated with the peptides, prodrugs, and pharmaceutical compositionsdisclosed herein may have cancer or may be at risk for developingcancer, including cancers characterized by solid tumors that may betreated with anti-angiogenic agents and/or anti-tumorigenic agents. Thepresently disclosed peptides, prodrugs, and pharmaceutical compositionsmay be utilized to treat cancers or hyperproliferative disorders, whichmay include, but are not limited to adenocarcinoma, melanoma, sarcoma,and teratocarcinoma and particularly cancers of the adrenal gland,bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, prostate, skin, testis, thymus, anduterus.

A “subject in need thereof” may include a subject having or at risk fordeveloping an eye disease. The presently disclosed peptides, prodrugs,and pharmaceutical compositions may be utilized to treat eye diseasesthat are characterized by neovascular retinal disease and that may betreated with anti-angiogenic agents, such as macular degeneration. Thepresently disclosed peptides, prodrugs, and pharmaceutical compositionsmay be utilized to treat eye diseases such as diabetic retinopathy orretinopathy of prematurity. The presently disclosed peptides, prodrugs,and pharmaceutical compositions may be utilized to treat eye diseases byadministering the disclosed peptide, prodrugs by intravitreal injection.

Macular degeneration is a medical condition predominantly found inelderly adults in which the center of the retina of the eye, otherwiseknown as the “macula” area of the retina, exhibits thinning, atrophy,and sometimes new blood vessel formation. Although macular degenerationsometimes may affect younger individuals, the term generally refers to“age-related” macular degeneration (i.e., “AMD” or “ARMD”). Advanced AMDhas two forms referred to as the “dry” and “wet” forms. The dry form ofadvanced AMD is characterized by central geographic atrophy, whichcauses vision loss through the loss of photoreceptors in the centralpart of the eye (i.e., rods and cones). The wet form of advanced AMD,otherwise referred to as “neovascular” or “exudative” AMD, causes visionloss due to abnormal blood vessel growth in the choriocapillaris,through a retinal layer referred to as “Bruch's membrane.” The wet formof AMD ultimately leads to blood and protein leakage below the macula.This bleeding, leaking, and scarring below the macula eventually causeirreversible damage to the photoreceptors and rapid vision loss if leftuntreated. Until recently, no effective treatments were known for wetmacular degeneration. However, new drugs that inhibit angiogenesis(i.e., “anti-angiogenic agents”) have been shown to cause regression ofthe abnormal blood vessels and improvement of vision. In order to beeffective, anti-angiogenic agents must be injected directly into thevitreous humor of the eye. The duration of effectiveness of suchinjections is impractically short for small peptides unless the latterare continuously released from a carrier macromolecule, microparticle,or nanoparticle, for which ester linkage provides controlled rates ofrelease.

The disclosed peptides and compositions thereof may be used in methodsfor treating or preventing other types of neovascular eye diseases,which may include neovascular eye diseases confined to the anteriorportion of the eye, such as the cornea or iris. Infectious andnon-infectious keratitis causes corneal clouding by formation ofnumerous empty neo-capillaries, this is often blinding and no adequatetreatments exists beyond corneal transplant. Many larger polypeptides orsmall charged molecules cannot penetrate corneal outer layers to reachthe capillary growth zones. Peptides described here have molecularweights under 1,500 and some have zero or only +1 net charge. With highpotency, these can penetrate the cornea topically from eye drops andmitigate corneal clouding.

A “subject in need thereof” may include a subject having or at risk fordeveloping a metabolic disease or disorder such as diabetes. The subjectmay have diabetes and may benefit from selective cytoprotection ofkidney cells from high glucose levels.

A “subject in need thereof” may include a subject having or at risk fordeveloping a kidney disease or disorder. The subject may have diabetesand may benefit from selective cytoprotection of kidney cells from highglucose levels. The subject may be undergoing chemotherapy (e.g., astreatment for cancer) and may benefit from selective cytoprotection ofkidney cells from genotoxic stress due to the chemotherapy.

A “subject in need thereof” may include a subject having or at risk fordeveloping an ear disease or disorder. The subject may be undergoingchemotherapy (e.g., as treatment for cancer) and may benefit fromtreatment and/or prevention of cytotoxic side effects of thechemotherapy.

A “subject in need thereof” may include a subject having or at risk fordeveloping an inflammatory disease or disorder (e.g., chronicinflammatory bowel diseases or disorders such as inflammatory boweldisease). The subject may have diabetes and/or may be undergoingchemotherapy and may benefit from a reduction in reactive oxygen species(ROS) in response to hyperglycemia and chemotherapy.

As used herein, the term “peptide” refers to a polymer of amino acidresidues joined by amide linkages. The term “amino acid residue,”includes but is not limited to amino acid residues contained in thegroup consisting of alanine (Ala or A), cysteine (Cys or C), asparticacid (Asp or D), glutamic acid (Glu or E), phenylalanine (Phe or F),glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), lysine(Lys or K), leucine (Leu or L), methionine (Met or M), asparagine (Asnor N), proline (Pro or P), glutamine (Gln or Q), arginine (Arg or R),serine (Ser or S), threonine (Thr or T), valine (Val or V), tryptophan(Trp or W), and tyrosine (Tyr or Y) residues. The term “amino acidresidue” also may include amino acid residues contained in the groupconsisting of homocysteine, 2-Aminoadipic acid, N-Ethylasparagine,3-Aminoadipic acid, Hydroxylysine, β-alanine, β-Amino-propionic acid,allo-Hydroxylysine acid, 2-Aminobutyric acid, 3-Hydroxyproline,4-Aminobutyric acid, 4-Hydroxyproline, piperidinic acid, 6-Aminocaproicacid, Isodesmosine, 2-Aminoheptanoic acid, allo-Isoleucine,2-Aminoisobutyric acid, N-Methylglycine, sarcosine, 3-Aminoisobutyricacid, N-Methylisoleucine, 2-Aminopimelic acid, 6-N-Methyllysine,2,4-Diaminobutyric acid, N-Methylvaline, Desmosine, Norvaline,2,2′-Diaminopimelic acid, Norleucine, 2,3-Diaminopropionic acid,Ornithine, and N-Ethylglycine. Typically, the amide linkages of thepeptides are formed from an amino group of the backbone of one aminoacid and a carboxyl group of the backbone of another amino acid.

As used herein, a peptide is defined as a short polymer of amino acids,of a length typically of 20 or less amino acids, and more typically of alength of 12 or less amino acids (Garrett & Grisham, Biochemistry,2^(nd) edition, 1999, Brooks/Cole, 110). In some embodiments, a peptideas contemplated herein may include no more than about 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. Apolypeptide, also referred to as a protein, is typically of length≥100amino acids (Garrett & Grisham, Biochemistry, 2^(nd) edition, 1999,Brooks/Cole, 110).

The peptides disclosed herein may be modified to include non-amino acidmoieties. Modifications may include but are not limited to carboxylation(e.g., N-terminal carboxylation via addition of a di-carboxylic acidhaving 4-7 straight-chain or branched carbon atoms, such as glutaricacid, succinic acid, adipic acid, and 4,4-dimethylglutaric acid),amidation (e.g., C-terminal amidation via addition of an amide orsubstituted amide such as alkylamide or dialkylamide), PEGylation (e.g.,N-terminal or C-terminal PEGylation via additional of polyethyleneglycol), acylation (e.g., O-acylation (esters), N-acylation (amides),S-acylation (thioesters)), acetylation (e.g., the addition of an acetylgroup, either at the N-terminus of the protein or at lysine residues),formylation lipoylation (e.g., attachment of a lipoate, a C8 functionalgroup), myristoylation (e.g., attachment of myristate, a C14 saturatedacid), palmitoylation (e.g., attachment of palmitate, a C16 saturatedacid), alkylation (e.g., the addition of an alkyl group, such as anmethyl at a lysine or arginine residue), isoprenylation or prenylation(e.g., the addition of an isoprenoid group such as farnesol orgeranylgeraniol), amidation at C-terminus, glycosylation (e.g., theaddition of a glycosyl group to either asparagine, hydroxylysine,serine, or threonine, resulting in a glycoprotein). Distinct fromglycation, which is regarded as a nonenzymatic attachment of sugars,polysialylation (e.g., the addition of polysialic acid), glypiation(e.g., glycosylphosphatidylinositol (GPI) anchor formation,hydroxylation, iodination (e.g., of thyroid hormones), andphosphorylation (e.g., the addition of a phosphate group, usually toserine, tyrosine, threonine or histidine).

The disclosed peptides may exhibit one or more biological functionsincluding anti-angiogenic activity, anti-tumorigenic activity,cytoprotective activity, mitigation of cytotoxicity (e.g., duringchemotherapy), and anti-inflammatory activity (e.g., via a reduction inreactive oxygen species (ROS)). Methods for measuring anti-angiogenicactivity, anti-tumorigenic activity, cytoprotective activity, mitigationof cytotoxicity, and reduction in reactive oxygen species (ROS) aredisclosed herein and are known in the art.

The disclosed peptides may be synthesized by any technique known tothose of skill in the art and by methods as disclosed herein. Methodsfor synthesizing the disclosed peptides may include chemical synthesisof proteins or peptides, the expression of peptides through standardmolecular biological techniques, and/or the isolation of proteins orpeptides from natural sources. The disclosed peptides thus synthesizedmay be subject to further chemical and/or enzymatic modification.Various methods for commercial preparations of peptides and polypeptidesare known to those of skill in the art.

Reference is made herein to peptides (e.g., PEDF-peptides or variantsthereof), polypeptides and pharmaceutical compositions comprisingpeptides and polypeptides. Exemplary peptides and polypeptides maycomprise, consist essentially of, or consist of the amino acid sequenceof any of SEQ ID NOs:1-13, or variants of the peptides and polypeptidesmay comprise, consist essentially of, or consist of an amino acidsequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, or 99%sequence identity to any of SEQ ID NOs:1-13. Variant peptidespolypeptides may include peptides or polypeptides having one or moreamino acid substitutions, deletions, additions and/or amino acidinsertions relative to a reference peptide or polypeptide. Alsodisclosed are nucleic acid molecules that encode the disclosed peptidesand polypeptides (e.g., polynucleotides that encode the peptides orpolypeptide of any of SEQ ID NOs:1-13 and variants thereof).

The amino acid sequences contemplated herein may include conservativeamino acid substitutions relative to a reference amino acid sequence(e.g., relative to the wild-type human PEDF amino acid sequence. Forexample, a variant peptide or polypeptide as contemplated herein mayinclude conservative amino acid substitutions and/or non-conservativeamino acid substitutions relative to a reference peptide or polypeptide.“Conservative amino acid substitutions” are those substitutions that arepredicted to interfere least with the properties of the referencepolypeptide, and “non-conservative amino acid substitution” are thosesubstitutions that are predicted to interfere most with the propertiesof the reference polypeptide. In other words, conservative amino acidsubstitutions substantially conserve the structure and the function ofthe reference protein. The following table provides a list of exemplaryconservative amino acid substitutions.

TABLE 1 Original Residue Conservative Substution Ala Gly, Ser Arg His,Lys Asn Asp, Gln, His Asp Asn, Glu Cys Ala, Ser Gln Asn, Glu, His GluAsp, Gln, His Gly Ala His Asn, Arg, Gln, Glu Ile Leu, Val Leu Ile, ValLys Arg, Gln, Glu Met Leu, Ile Phe His, Met, Leu, Trp, Tyr Ser Cys, ThrThr Ser, Val Trp Phe, Tyr Tyr His, Phe, Trp Val Ile, Leu, Thr

Conservative amino acid substitutions generally maintain: (a) thestructure of the peptide or polypeptide backbone in the area of thesubstitution, for example, as a beta sheet or alpha helicalconformation, (b) the charge or hydrophobicity of the molecule at thesite of the substitution, and/or (c) the bulk of the side chain.Non-conservative amino acid substitutions generally disrupt: (a) thestructure of the peptide or polypeptide backbone in the area of thesubstitution, for example, as a beta sheet or alpha helicalconformation, (b) the charge or hydrophobicity of the molecule at thesite of the substitution, and/or (c) the bulk of the side chain.

Also contemplated herein are peptidomimetics of the disclosed peptides.As disclosed herein, a peptidomimetic is a peptide equivalentcharacterized as retaining the polarity, three dimensional size andfunctionality (bioactivity) of its peptide equivalent but where thepeptide bonds have been replaced (e.g., by more stable linkages whichare more resistant to enzymatic degradation by hydrolytic enzymes).Generally, the bond which replaces the amide bond conserves many of theproperties of the amide bond (e.g., conformation, steric bulk,electrostatic character, and possibility for hydrogen bonding). Ageneral discussion of prior art techniques for the design and synthesisof peptidomimetics is provided in “Drug Design and Development”, Chapter14, Krogsgaard, Larsen, Liljefors and Madsen (Eds) 1996, Horwood Acad.Pub, the contents of which are incorporated herein by reference in theirentirety. Suitable amide bond substitutes include the following groups:N-alkylation (Schmidt, R. et. al., Int. J. Peptide Protein Res., 1995,46, 47), retro-inverse amide (Chorev, M and Goodman, M., Acc. Chem. Res,1993, 26, 266), thioamide (Sherman D. B. and Spatola, A. F. J. Am. Chem.Soc., 1990, 112, 433), thioester, phosphonate, ketomethylene (Hoffman,R. V. and Kim, H. O. J. Org. Chem., 1995, 60, 5107), hydroxymethylene,fluorovinyl (Allmendinger, T. et al., Tetrahydron Lett., 1990, 31,7297), vinyl, methyleneamino (Sasaki, Y and Abe, J. Chem. Pharm. Bull.1997 45, 13), methylenethio (Spatola, A. F., Methods Neurosci, 1993, 13,19), alkane (Lavielle, S. et. al., Int. J. Peptide Protein Res., 1993,42, 270) and sulfonamido (Luisi, G. et al. Tetrahedron Lett. 1993, 34,2391), which all are incorporated herein by reference in theirentireties. The peptides and polypeptide disclosed herein may includepeptidomimetic equivalents.

Variants comprising deletions relative to a reference amino acidsequence are contemplated herein. A “deletion” refers to a change in theamino acid or nucleotide sequence that results in the absence of one ormore amino acid residues or nucleotides relative to a referencesequence. A deletion removes at least 1, 2, 3, 4, 5, 10, 20, 50, 100, or200 amino acids residues or nucleotides. A deletion may include aninternal deletion or a terminal deletion (e.g., an N-terminal truncationor a C-terminal truncation of a reference polypeptide or a 5′-terminalor 3′-terminal truncation of a reference polynucleotide).

Variants comprising fragment of a reference amino acid sequence arecontemplated herein. A “fragment” is a portion of an amino acid sequenceor a polynucleotide which is identical in sequence to but shorter inlength than a reference sequence. A fragment may comprise up to theentire length of the reference sequence, minus at least onenucleotide/amino acid residue. For example, a fragment may comprise from5 to 1000 contiguous nucleotides or contiguous amino acid residues of areference polynucleotide or reference polypeptide, respectively. In someembodiments, a fragment may comprise at least 5, 10, 15, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 150, 250, or500 contiguous nucleotides or contiguous amino acid residues of areference polynucleotide or reference polypeptide, respectively.Fragments may be preferentially selected from certain regions of amolecule. The term “at least a fragment” encompasses the full lengthpolynucleotide or full length polypeptide.

Variants comprising insertions or additions relative to a referencesequence are contemplated herein. The words “insertion” and “addition”refer to changes in an amino acid or sequence resulting in the additionof one or more amino acid residues. An insertion or addition may referto 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, or 200amino acid residues.

Fusion proteins also are contemplated herein. The disclosed polypeptidesmay comprise fusion proteins. A “fusion protein” refers to a proteinformed by the fusion of at least one peptide or variant thereof asdisclosed herein to at least one molecule of a heterologous protein (orfragment or variant thereof). The heterologous protein(s) may be fusedat the N-terminus, the C-terminus, or both termini of the peptides orvariants thereof. A fusion protein comprises at least a fragment orvariant of the heterologous protein and at least a fragment or variantof the presently disclosed peptides, which are associated with oneanother, preferably by genetic fusion (i.e., the fusion protein isgenerated by translation of a nucleic acid in which a polynucleotideencoding all or a portion of the heterologous protein is joined in-framewith a polynucleotide encoding all or a portion of the disclosedpeptides or variants thereof). The heterologous protein and peptide,once part of the fusion protein, may each be referred to herein as a“portion”, “region” or “moiety” of the fusion protein (e.g., a “aheterologous protein portion” or a “PEDF peptide portion”).

“Homology” refers to sequence similarity or, interchangeably, sequenceidentity, between two or more polypeptide sequences. Homology, sequencesimilarity, and percentage sequence identity may be determined usingmethods in the art and described herein.

The phrases “percent identity” and “% identity,” as applied topolypeptide sequences, refer to the percentage of residue matchesbetween at least two polypeptide sequences aligned using a standardizedalgorithm. Methods of polypeptide sequence alignment are well-known.Some alignment methods take into account conservative amino acidsubstitutions. Such conservative substitutions, explained in more detailabove, generally preserve the charge and hydrophobicity at the site ofsubstitution, thus preserving the structure (and therefore function) ofthe polypeptide. Percent identity for amino acid sequences may bedetermined as understood in the art. (See, e.g., U.S. Pat. No.7,396,664, which is incorporated herein by reference in its entirety). Asuite of commonly used and freely available sequence comparisonalgorithms is provided by the National Center for BiotechnologyInformation (NCBI) Basic Local Alignment Search Tool (BLAST) (Altschul,S. F. et al. (1990) J. Mol. Biol. 215:403 410), which is available fromseveral sources, including the NCBI, Bethesda, Md., at its website. TheBLAST software suite includes various sequence analysis programsincluding “blastp,” that is used to align a known amino acid sequencewith other amino acids sequences from a variety of databases.

Percent identity may be measured over the length of an entire definedpolypeptide sequence, for example, as defined by a particular SEQ IDnumber, or may be measured over a shorter length, for example, over thelength of a fragment taken from a larger, defined polypeptide sequence,for instance, a fragment of at least 15, at least 20, at least 30, atleast 40, at least 50, at least 70 or at least 150 contiguous residues.Such lengths are exemplary only, and it is understood that any fragmentlength supported by the sequences shown herein, in the tables, figuresor Sequence Listing, may be used to describe a length over whichpercentage identity may be measured.

A “variant” of a particular polypeptide sequence may be defined as apolypeptide sequence having at least 50% sequence identity to theparticular polypeptide sequence over a certain length of one of thepolypeptide sequences using blastp with the “BLAST 2 Sequences” toolavailable at the National Center for Biotechnology Information'swebsite. (See Tatiana A. Tatusova, Thomas L. Madden (1999), “Blast 2sequences—a new tool for comparing protein and nucleotide sequences”,FEMS Microbiol Lett. 174:247-250). Such a pair of polypeptides may show,for example, at least 60%, at least 70%, at least 80%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% or greatersequence identity over a certain defined length of one of thepolypeptides. A “variant” may have substantially the same functionalactivity as a reference polypeptide. For example, a variant may exhibitor more biological activities associated with PEDF. “Substantiallyisolated or purified” nucleic acid or amino acid sequences arecontemplated herein. The term “substantially isolated or purified”refers to nucleic acid or amino acid sequences that are removed fromtheir natural environment, and are at least 60% free, preferably atleast 75% free, and more preferably at least 90% free, even morepreferably at least 95% free from other components with which they arenaturally associated.

A “composition comprising a given polypeptide” refers broadly to anycomposition containing the given amino acid sequence. The compositionmay comprise a dry formulation or an aqueous solution. The compositionsmay be stored in any suitable form including, but not limited to,freeze-dried form and may be associated with a stabilizing agent such asa carbohydrate. The compositions may be aqueous solution containingsalts (e.g., NaCl), detergents (e.g., sodium dodecyl sulfate; SDS), andother components (e.g., Denhardt's solution, dry milk, salmon sperm DNA,and the like).

The disclosed pharmaceutical composition may comprise the disclosedpeptides, polypeptides, variants at any suitable dose. Suitable dosesmay include, but are not limited to, about 0.01 μg/dose, about 0.05μg/dose, about 0.1 μg/dose, about 0.5 μg/dose, about 1 μg/dose, about 2μg/dose, about 3 μg/dose, about 4 μg/dose, about 5 μg/dose, about 10μg/dose, about 15 μg/dose, about 20 μg/dose, about 25 μg/dose, about 30μg/dose, about 35 μg/dose, about 40 μg/dose, about 45 μg/dose, about 50μg/dose, about 100 μg/dose, about 200 μg/dose, about 500 μg/dose, orabout 1000 μg/dose.

The disclosed peptides, polypeptides, or variants thereof may beadministered at any suitable dose level. In some embodiments, a subjectin need thereof is administered a peptide, polypeptide, or variantthereof at a dose level of from about 1 ng/kg up to about 2000 ng/kg. Insome embodiments, the peptide, polypeptide, or variant thereof isadministered to the subject in need thereof at a dose level of at leastabout 1 ng/kg, 2 ng/kg, 5 ng/kg, 10 ng/kg, 20 ng/kg, 50 ng/kg, 100ng/kg, 200 ng/kg, 500 ng/kg, 1000 ng/kg or 2000 ng/kg. In otherembodiments, the peptide, polypeptide, or variant thereof isadministered to the subject in need thereof at a dose level of less thanabout 2000 ng/kg, 1000 ng/kg, 500 ng/kg, 200 ng/kg, 100 ng/kg, 50 ng/kg,20 ng/kg, 10 ng/kg, 5 ng/kg, 2 ng/kg, or 1 ng/kg. In furtherembodiments, the peptide, polypeptide, or variant thereof isadministered to a subject in need thereof within a dose level rangebounded by any 1 ng/kg, 2 ng/kg, 5 ng/kg, 10 ng/kg, 20 ng/kg, 50 ng/kg,100 ng/kg, 200 ng/kg, 500 ng/kg, 1000 ng/kg or 2000 ng/kg.

The disclosed peptides, polypeptides, or variants thereof may beadministered under any suitable dosing regimen. Suitable dosing regimensmay include, but are not limited to, daily regimens (e.g., 1 dose/dayfor 1, 2, 3, 4, 5, 6, 7 or more days), twice daily regimens (e.g., 2doses/day for 1, 2, 3, 4, 5, 6, 7 or more days), and thrice dailyregiments (e.g., 3 doses/day for 1, 2, 3, 4, 5, 6, 7 or more days).Suitable regiments also may include dosing every other day, 3times/week, once a week, for 1, 2, 3, 4, or more weeks.

The peptides and prodrugs utilized in the methods disclosed herein maybe formulated as a pharmaceutical composition for delivery via anysuitable route (e.g. parenteral or intravitreal routes). As such,pharmaceutical compositions comprising the peptides and prodrugs may beadapted for administration by any appropriate route, for exampleintravitreal or intraperitoneal, with the intention of localconfinement, and parenteral (including subcutaneous, intramuscular,intravenous or intradermal) route. Such formulations may be prepared byany method known in the art of pharmacy, for example by bringing intoassociation the active ingredient with suitable carrier(s) orexcipient(s). In some embodiments, the prodrug carrier-bound formsdescribed herein may be intended for less frequent dosing ranging fromonce weekly to once monthly to once per 2 months or less frequently.Pharmaceutical compositions adapted for parenteral administrationinclude aqueous and non-aqueous sterile injection solutions which maycontain anti-oxidants, buffers, bacteriostats and solutes which renderthe formulation isotonic with bodily fluid of the intended recipient;and aqueous and non-aqueous sterile suspensions which may includesuspending agents and thickening agents. The formulations may bepresented in unit-dose or multi-dose containers, for example sealedampoules and vials, and may be stored in a freeze-dried (lyophilized)condition requiring only the addition of the sterile liquid carrier, forexample water for injections, immediately prior to use.

Anti-Angiogenic and Anti-Tumorigenic Peptides

The disclosed peptides may be described as modified peptides comprisingan N-terminal carboxyl group. The modified peptides disclosed herein maybe described as having a modified amino acid sequence as follows:Z-B-X-AA0-AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9-AA10-Y  (SEQ ID NO:14)

where:

AA0, AA1, AA2, AA3, AA4, AA5, AA6, AA7, AA8, and AA9 are selected from anaturally occurring amino acid, including sarcosine, and beta-alanine;

AA0 is absent or present, and when AA0 is present AA0 is selected fromsarcosyl and Gly; or beta-alanyl;

AA1 is absent or present, and when AA1 is present AA1 is selected fromsarcosyl and Gly;

AA2 is absent or present, and when AA2 is present AA2 optionally is Tyr;

AA3 is Asp or Asn, optionally when AA2 is present;

AA4 is Leu or Val;

AA4 is Leu;

AA5 is Tyr or Phe;

AA6 is Arg;

AA7 is Val or Pro;

AA8 is present or absent, and when AA8 is present AA8 is selected fromArg, Pro, and Gln;

AA9 is present or absent, and when AA9 is present AA9 selected from Serand Ala;

AA10 is absent or present and selected from Ser, Ser-Thr, Ser-Thr-Ser,Ser-Thr-Ser-Pro, and Ser-Thr-Ser-Pro-Thr;

X is absent or present, and when X is present X is selected from thegroup consisting of acetyl, butyryl, hexanoyl, methoxy-PEG_((n))CO,hydroxy-PEG_((n))CO, amino-PEG_((n))CO, Of andsarcosyl-amino-PEG_((n))CO, where n is 3-13, and X is in an amide bondto an amino terminus of AA0, AA1, or AA2;

B is a di-carboxylic acid containing from 4-8 carbon atoms, which may bestraight-chain or branched, and B is in a half-amide bond to an aminoterminus of X, or B is in a half-amide bond to an amino terminus of AA0when X is absent, or B is in a half-amide bond to an amino terminus ofAA1 when X and AA0 are absent, or B is in a half-amide bond to an aminoterminus of AA2 when X, AA0 and AA1 are absent, or B is in a half-amidebond to an amino terminus of AA3 when X, AA0, AA1, and AA2 are absent,and B may end in a free carboxyl group, or this otherwise free carboxylgroup may be in an ester bond to a hydroxyl group of Z;

Z is absent or present, and when Z is present Z is selected from thegroup consisting of: primary or secondary hydroxyl groups of sugarmonomers present on a polymeric carbohydrate carrier; hydroxyl groups ofa hydroxyl terminal dendrimer; and primary or secondary hydroxyl groupsof acyclic or cyclic amino alcohols having between 4 and 6 carbon atomsand a single primary or secondary amine that is protected through anamide bond or a carbamate bond; and Y is an amide or a substituted amideselected from alkylamide, dialkylamide, and PEG_((n))-amide, where n is4-12.

The disclosed modified peptides typically include a free N-terminalcarboxyl group. For example, the modified peptides may be formed byadding a di-carboxylic acid to an N-terminal amino group to form ahalf-amide bond. Suitable di-carboxylic acids may include, but are notlimited to di-carboxylic acids comprising 4-7 straight-chain or branchedcarbon atoms such as glutaric acid, succinic acid, adipic acid, and4,4-dimethylglutaric acid.

In some embodiments, the modified peptide may comprise a modified aminoacid sequence as follows: B-X-AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9-Y (SEQID NO:14), where X, AA1, AA2, AA3, AA4, AA5, AA6, AA7, AA8, AA9, and Yare as defined above. In other embodiments, the modified peptide maycomprise a modified amino acid sequence as follows:B-sarcosyl-AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9-Y (SEQ ID NO:14), whereB, AA1, AA2, AA3, AA4, AA5, AA6, AA7, AA8, AA9, and Y are as definedabove. In further embodiments, the modified peptide may comprise amodified amino acid sequence as follows:Z-B-sarcosyl-AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9-amide (SEQ ID NO:14),where AA1, AA2, AA3, AA4, AA5, AA6, AA7, AA8, and AA9 are as definedabove. In even further embodiments, the modified peptide may comprise amodified amino acid sequence as follows: theglutaryl-sarcosyl-Gly-Tyr-AA3-Leu-Tyr-Arg-Val-AA8-AA9-amide (SEQ IDNO:14), where AA3, AA8, and AA9 are as defined above.

In some embodiments, the modified peptide may comprise an N-terminalgroup selected from glutaryl; adipoyl, suberoyl,4,4-dimetlyglutaryl-Sar; adipoyl-Sar-PEG(4-12);4,4-dimetlyglutaryl-Sar-PEG(4-12)-Sar; or PEG-(4-13)-CO). In otherembodiments, the modified peptide may comprise a modified amino acidsequence having a N-terminal group selected from glutaryl-sarcosyl or4,4-dimetlyglutaryl-sarcosyl. In further embodiments, the modifiedpeptide may comprise a modified amino acid sequence as follows:glutaryl-sarcosyl-AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9-amide (SEQ IDNO:14) or adipoyl-sarcosyl-AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9-amide(SEQ ID NO:14), oradipoyl-sarcosyl-AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9-amide (SEQ ID NO:14),or suberoyl-AA3-AA4-AA5-AA6-AA7-AA8-AA9-amide (SEQ ID NO:14) where AA1,AA2, AA3, AA4, AA5, AA6, AA7, AA8, and AA9 are as defined above.

Specifically, the disclosed peptides may include modified peptidescomprising a modified amino acid sequence selected from:glutaryl-sarcosyl-Gly-Tyr-Asp-Leu-Tyr-Arg-Val-Arg-Ser-amide (i.e., SEQID NO:2); andglutaryl-sarcosyl-Gly-Tyr-Asn-Leu-Tyr-Arg-Val-Arg-Ser-amide (i.e., SEQID NO:4), where a charged amino acid in a reference sequence (e.g.,PEDF) is replaced with a neutral amino acid(sarcosyl-NH-PEG(4)CO-Sar-Tyr-Asn-Leu-Tyr-Arg-Val-Arg-Ser-amide (i.e.,SEQ ID NO:6)). In addition, the disclosed peptides also includetruncation and modifications near and at the C-terminus as inglutaryl-sarcosyl-Tyr-Asn-Leu-Tyr-Arg-Val-Pro-ethylamide (SEQ ID NO:8)(see also SEQ ID NO:10 and SEQ ID NO:12), where a second charged Argresidue is also replaced by a neutral one, Pro, yielding a smaller, morepotent peptide, andglutaryl-sarcosyl-Tyr-Asn-Leu-Tyr-Arg-Val-Gln-Ser-amide (SEQ ID NO:9)where the Arg is replaced by Gln, without truncation, again withenhanced potency.

Prodrugs of the disclosed peptides also are contemplated herein. In someembodiments, prodrugs of the disclosed peptides may comprise any of thedisclosed peptides and a microparticle carrier or nanoparticle carrierhaving a free hydroxyl, where the N-terminal carboxyl group of thepeptide is esterified to the free hydroxyl group of the carrier in orderto form a carrier prodrug of the disclosed peptides.

In other embodiments of the prodrugs, the prodrugs may comprise: (a) anyof the disclosed peptides; (b) a linker having a free hydroxyl group anda free amino group (e.g., an amino alcohol linker) for linking thepeptides to the carriers (i.e., microparticle carriers and/ornanoparticle carriers); and (c) a carrier having a free hydroxyl groupor a free carboxyl group. In this embodiment, the prodrug is prepared by(a) esterifying the peptide to the free hydroxyl group of the linker;and by (b) attaching the linker to the carrier via the amino group ofthe linker and a free hydroxyl group of the carrier to form a carbamatebond, or by attaching the amino group of the linker and a free carboxylgroup of the carrier to form an amide bond. Suitable linkers mayinclude, but are not limited to amino-n-butoxy, amino-ethoxyethyloxy,amino-piperidyl(3, or 4)-oxy, amino-pyrrolidinyl(3)-oxy,amino-benzyloxy, BOC-aminoethylamido-valeric acid (4)-oxy,amino-cyclohexyl (3, or 4)-oxy, and amino-cyclopentyl (3)-oxy.

The carriers of the prodrug may be microparticle carriers and/ornanoparticle carriers. For example, the carriers of the prodrug may havean average effective diameter that is less than about 0.2 microns (e.g.,when the carriers are formulated for intravitreal administration). Insome embodiments, the carriers of the prodrug may have an averageeffective diameter that ranges from 0.05 microns to 20 microns (e.g.,when the carriers are formulated for intraperitoneal administration). Insome embodiments, the carriers are nanoparticle carriers having anaverage effective diameter that is less than about 1 micron (e.g., anaverage effective diameter that ranges from 0.01 microns to 1 micron).The carrier typically comprises a polymeric material having terminalhydroxyl groups and/or terminal carboxyl groups. In some embodiments,the carriers comprise dendrimers having terminal hydroxyl groups and/orterminal carboxyl groups. In further embodiments, the carriers comprisedextran and/or hyaluronic acid, and optionally the dextran or hyaluronicacid is crosslinked and/or condensed. Optionally, the carriers may beoptically transparent or substantially optically transparent (e.g., whenthe carriers are used in preparing a prodrug for intravitrealadministration). For example, a transparent or substantially transparentcarriers may absorb and reflect less than about 10%, 9%, 8%, 7%, 6%, 5%,4%, 3%, 2%, or 1% of incident light and/or may have a totaltransmittance of at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% of incident light.

The modified peptides disclosed herein and the prodrugs thereof may beformulated as pharmaceutical compositions for use in treating and/orpreventing diseases or disorders that are amenable to treatment byanti-angiogenic agents and/or anti-tumorigenic agents. As such, thepharmaceutical compositions may be administered to a subject in order toinhibit angiogenesis and/or tumorigenesis in the subject.

The disclosed methods may include administering to a subject aneffective amount of a peptide, prodrug, or pharmaceutical composition totreat and/or prevent a disease and/or disorder. As used herein, thephrase “effective amount” shall mean that drug dosage that provides thespecific pharmacological response for which the drug is administered ina significant number of subjects in need of such treatment. An effectiveamount of a drug that is administered to a particular subject in aparticular instance will not always be effective in treating theconditions/diseases described herein, even though such dosage is deemedto be a therapeutically effective amount by those of skill in the art.

The disclosed methods may include administering to a subject aneffective amount of a peptide, prodrug, or pharmaceutical compositionfor inhibiting angiogenesis and/or tumorigenesis relative to a control.In some embodiments, angiogenesis and/or tumorigenesis is inhibited byat least 10%, at least 25%, at least 50%, at least 75%, at least 90%, orat least 95% in a treated sample relative to an untreated controlsample.

ILLUSTRATIVE EMBODIMENTS

The following embodiments are illustrative and should not be interpretedto limit the scope of the claimed subject matter.

Embodiment 1

A modified peptide comprising an N-terminal carboxyl group and amodified amino acid sequence as follows:Z-B-X-AA0-AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9-AA10-Y  (SEQ ID NO:14)wherein:

-   AA0, AA1, AA2, AA3, AA4, AA5, AA6, AA7, AA8, and AA9 are selected    from a naturally occurring amino acid, including sarcosine, and beta    alanine;-   AA0 is absent or present, and when AA0 is present AA0 is selected    from sarcosyl and Gly; or beta alaninyl;-   AA1 is absent or present, and when AA1 is present AA1 is selected    from sarcosyl and Gly;-   AA2 is Tyr;-   AA2 is absent or present, and when AA2 is present AA3 is selected    from Asp or Asn;-   AA3 is Asp or Asn;-   AA4 is Leu or Val;-   AA5 is Tyr or Phe;-   AA6 is Arg;-   AA7 is Val or Pro;-   AA8 is present or absent, and when AA8 is present AA8 is selected    from Arg, Pro, and Gln;-   AA9 is present or absent, and when AA9 is present AA9 selected from    Ser and Ala;-   AA10 is absent or present and selected from Ser, Ser-Thr,    Ser-Thr-Ser, Ser-Thr-Ser-Pro, and Ser-Thr-Ser-Pro-Thr;-   X is absent or present, and when X is present X is selected from the    group consisting of acetyl, butyryl, hexanoyl, methoxy-PEG_((n))CO,    hydroxy-PEG_((n))CO, amino-PEG_((n))CO, and    sarcosyl-amino-PEG_((n))CO, where n is 3-13, and X is in an amide    bond to an amino terminus of AA0, AA1, or AA2;-   B is a di-carboxylic acid containing from 4-8 carbon atoms, which    may be straight-chain or branched, and B is in a half-amide bond to    an amino terminus of X, or B is in a half-amide bond to an amino    terminus of AA0 when X is absent, or B is in a half-amide bond to an    amino terminus of AA1 when X and AA0 are absent, or B is in a    half-amide bond to an amino terminus of AA2 when X, AA0 and AA1 are    absent, or B is in a half-amide bond to an amino terminus of AA3    when X, AA0, AA1, and AA2 are absent; and B may end in a free    carboxyl group, or this otherwise free carboxyl group may be in an    ester bond to a hydroxyl group of Z;-   Z is absent or present, and when Z is present Z is selected from the    group consisting of: primary or secondary hydroxyl groups of sugar    monomers present on a polymeric carbohydrate carrier; hydroxyl    groups of a hydroxyl terminal dendrimer; and primary or secondary    hydroxyl groups of acyclic or cyclic amino alcohols having between 4    and 6 carbon atoms and a single primary or secondary amine that is    protected through an amide bond or a carbamate bond; and-   Y is an amide or a substituted amide selected from alkylamide,    dialkylamide, and PEG_((n))-amide, where n is 4-12.

Embodiment 2

The modified peptide of embodiment 1 comprising a modified amino acidsequence as follows: B-AA0-AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9-Y (SEQ IDNO:14).

Embodiment 3

The modified peptide of embodiment 1 or 2, wherein B is adipic acid orglutaric acid in half amide bond to AA0, or B is in a half-amide bond toan amino terminus of AA1 when AA0 is absent, or B is in a half-amidebond to an amino terminus of AA2 when AA0 and AA1 are absent.

Embodiment 4

The modified peptide of any of the foregoing embodiments whereinAA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9 is Tyr-Asn-Leu-Tyr-Arg-Val-Arg-Ser (SEQID NO:14).

Embodiment 5

The modified peptide of any of the foregoing embodiments whereinAA2-AA3-AA4-AA5-AA6 is Tyr-Asn-Leu-Tyr-Arg (SEQ ID NO:14).

Embodiment 6

The modified peptide of any of the foregoing embodiments wherein AA7 isVal or Pro-ethylamide when AA6 is the C-terminal amino acid of thepeptide.

Embodiment 7

The modified peptide of any of the foregoing embodiments wherein AA7 isVal and AA8 is Pro-ethylamide when AA8 is the C-terminal amino acid ofthe peptide.

Embodiment 8

The modified peptide of any of the foregoing embodiments whereinAA7-AA8-AA9 is Val-Gln-Ser.

Embodiment 9

The modified peptide of any of the foregoing embodiments wherein Y isamide or ethylamide.

Embodiment 10

The modified peptide of any of the foregoing embodiments wherein thepeptide does not have an Arg at position AA8.

Embodiment 11

The modified peptide of any of the foregoing embodiments having aN-terminal moiety, which may be substituent B, selected from succinicacid, glutaric acid, adipic acid, and suberic acid.

Embodiment 12

The modified peptide of any of the foregoing embodiments comprising amodified amino acid sequence as follows:sarcosyl-AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9-Y (SEQ ID NO:14).

Embodiment 13

The modified peptide of any of the foregoing embodiments comprising amodified amino acid sequence as follows:glutaryl-sarcosyl-AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9-amide (SEQ IDNO:14).

Embodiment 14

The modified peptide of any of the foregoing embodiments comprising amodified amino acid sequence as follows:glutaryl-sarcosyl-Gly-Tyr-AA3-Leu-Tyr-Arg-Val-AA8-AA9-amide (SEQ IDNO:14).

Embodiment 15

The modified peptide of any of the foregoing embodiments comprising amodified amino acid sequence:glutaryl-sarcosyl-Gly-Tyr-Asp-Leu-Tyr-Arg-Val-Arg-Ser-amide (i.e., SEQID NO:2).

Embodiment 16

The modified peptide of any of the foregoing embodiments comprising amodified amino acid sequence:glutaryl-sarcosyl-Gly-Tyr-Asn-Leu-Tyr-Arg-Val-Arg-Ser-amide (i.e., SEQID NO:4).

Embodiment 17

The modified peptide of any of the foregoing embodiments comprising amodified amino acid sequence:adipoyl-sarcosyl-Tyr-Asn-Leu-Tyr-Arg-Val-Arg-Ser-amide (i.e., SEQ IDNO:11).

Embodiment 18

The modified peptide of any of the foregoing embodiments comprising amodified amino acid sequence:glutaryl-sarcosyl-Gly-Tyr-Asn-Leu-Tyr-Arg-Val-Pro-ethylamide (i.e., SEQID NO:8).

Embodiment 19

The modified peptide of any of the foregoing embodiments comprising amodified amino acid sequence:adipoyl-sarcosyl-Tyr-Asn-Leu-Tyr-Arg-Val-Pro-ethylamide (i.e., SEQ IDNO:10).

Embodiment 20

The modified peptide of any of the foregoing embodiments comprising amodified amino acid sequence disclosed in FIG. 1.

Embodiment 21

A prodrug of any of the foregoing modified peptides comprising amodified amino acid sequence as follows:Z-B-AA0-AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9-Y (SEQ ID NO:14) where Z isan amido or carbamato alcohol in ester bond linkage to B, and Z is inamide or carbamate bond linkage to a carboxyl group or a hydroxyl groupof a polymeric carrier particle.

Embodiment 22

A prodrug of any of the modified peptides of embodiments 1-20 in whichthe N-terminal carboxyl group of the peptide is esterified to a freehydroxyl group of a particulate carrier.

Embodiment 23

A prodrug of any of the modified peptides of embodiments 1-20 in whichthe prodrug further comprises an amino alcohol linker and the peptide isesterified to a free hydroxyl group of the linker, and the linker isattached to a particulate carrier via the amino group of the linker anda free hydroxyl group of the particulate carrier forming a carbamatebond or via the amino group of the linker and a free carboxyl group ofthe particulate carrier forming an amide bond, and optionally the linkeris selected from the group consisting of amino-n-butoxy,amino-ethoxyethyloxy, amino-piperidyl(3, or 4)-oxy,amino-pyrrolidinyl(3)-oxy, amino-benzyloxy, BOC-aminoethylamido-valericacid (4)-oxy, amino-cyclohexyl (3, or 4)-oxy, and amino-cyclopentyl(3)-oxy.

Embodiment 24

The prodrug of any of embodiments 21-23, wherein the particulate carriercomprises dendrimers having terminal hydroxyl or carboxyl groups.

Embodiment 25

The prodrug of any of embodiments 21-24, wherein the particulate carriercomprises dextrin, dextran, or hyaluronic acid.

Embodiment 26

The prodrug of embodiment 25, wherein the dextrin, dextran, orhyaluronic acid is crosslinked and/or condensed.

Embodiment 27

A pharmaceutical composition comprising the modified peptides of any ofembodiments 1-20 or prodrugs thereof, optionally as the prodrugs of anyof embodiments 21-26, together with a pharmaceutical acceptable carrier,diluent, or excipient.

Embodiment 28

A method of treating and/or preventing cancer in a subject in needthereof, the method comprising administering the composition ofembodiment 27 to the subject.

Embodiment 29

The method of embodiment 28, wherein the subject has ovarian cancer orcolon cancer and the pharmaceutical composition is administered byparenteral administration.

Embodiment 30

A method of treating and/or preventing cancer in a subject having immunesuppression characterized by elevated PD-L1, the method comprisingadministering the composition of embodiment 27 to the subject.

Embodiment 31

The method of embodiment 30, wherein the subject has cancer types knownto be responsive to immune stimulation therapy such as but not limitedto melanoma, non-small cell lung cancer, bladder cancer, renal cellcarcinoma, and oral squamous cell carcinoma, and the pharmaceuticalcomposition is administered by parenteral administration.

Embodiment 32

A method of treating and/or preventing infectious disease in a subjecthaving immune suppression characterized by elevated PD-L1, the methodcomprising administering the composition of embodiment 27 to thesubject.

Embodiment 33

A method of treating and/or preventing an eye disease or disorder in asubject in need thereof, the method comprising administering thecomposition of embodiment 27 to the subject.

Embodiment 34

The method of embodiment 33, wherein the eye disease is a neovascularretinal disease.

Embodiment 35

The method of embodiment 34, wherein the neovascular retinal disease ismacular degeneration.

Embodiment 36

The method of embodiment 33, wherein the eye disease or disorder isdiabetic retinopathy or retinopathy of prematurity.

Embodiment 37

The method of embodiment 33, wherein the pharmaceutical composition isformulated for topical administration as eye drops for treating corneal(keratitis), iris, or retinal neovascular disease.

Embodiment 38

A method of treating and/or preventing a kidney disease or disorder in asubject in need thereof, the method comprising administering thepharmaceutical composition of embodiment 27 to the subject.

Embodiment 39

The method of embodiment 38, wherein the subject has diabetes and themethod protects kidney cells from high glucose.

Embodiment 40

The method of embodiment 38, wherein the subject is undergoingchemotherapy and the method protects kidney cells from genotoxic stress.

Embodiment 41

A method of treating and/or preventing an ear disease or disorder in asubject in need thereof, the method comprising administering thepharmaceutical composition of embodiment 27 to the subject.

Embodiment 42

The method of embodiment 41, wherein the subject is undergoingchemotherapy and the method treats and/or prevents cytotoxicity.

Embodiment 43

Any 8-10 amino acid anti-angiogenic peptide with C-terminal amide orethylamide which is N-terminally capped as half-amide with adipic acid,which esterified to an amino alcohol, where the latter is furtherattached as a carbamate or amide to OH groups or carboxyl groups of apolymeric carrier, a specific example being:amino-alkoxy-adipoyl-Gly-Val-DalloI-Ser-Gln-Ile-Arg-Pro-ethylamide incarbamate linkage to a condensed dextran hydrogel.

EXAMPLES

The following Examples are illustrative and are not intended to limitthe scope of the claimed subject matter.

Example 1—Anti-Angiogenic PEDF Peptides and their Ester Pro-Drugs forTreating Cancer and Eye Diseases

Abstract

Cancer growth, and retina diseases that cause blindness are both drivenby excessive formation of the new capillaries (angiogenesis). Ovariancancer (OvCa) initially responds to chemotherapy but resistant cellsremain and re-grow. Chemotherapy for OvCa is typically administeredthrough intravenous (IV) route, but is more effective when deliveredlocally (intraperitoneally, IP). Adding an anti-angiogenic agentAvastin, which neutralizes vascular endothelial growth factor (VEGF),can slow relapse; however, it is not amenable to local IP delivery andis typically given IV. Longer remission caused by Avastin iscounterbalanced by its VEGF-related toxicities, which include intestinalperforation, and hypertension. Avastin and other anti-VEGF proteins arealso used to alleviate exudative macular degeneration, where excessivepathologic capillary sprouting beneath the retina destroys retinallayers. None of these proteins is suited for sustained releaseformulation, and their rapid clearance from the eye necessitatesburdensome monthly intraocular injections. We invented non-toxic smallanti-angiogenic peptides that mimic activity of the natural angiogenesisinhibitor Pigment Epithelium-Derived Factor (PEDF). We have previouslypatented an 18 amino-acid PEDF peptide, P18, which slows cancer growthand blocks ocular angiogenesis. (See U.S. Pat. Nos. 9,096,689;8,198,406; 7,723,313; 7,105,496; 6,919,309; and 6,797,691; the contentsof which are incorporated herein by reference in their entireties).

We now report, more potent, practical and safe, 8-10 amino acid peptidesrelated to or derived from P18, which inhibit angiogenesis and directlykill ovarian cancer cells. The disclosed peptides include significantchemical and amino acid sequence modifications. The same peptidesinhibit capillary sprouting from tissue explants prepared from mousechoroid; these results imply efficacy vs. eye disease. These peptidesare appended with novel linking groups to facilitate metastable chemicalattachment to nanoparticles for slow (>1 month) release for extendedtherapeutic effect when given IP, intra-cranially or intra-vitreally.Our new peptides may be conjugated to nanoparticles, which are designedto anchor at their site of delivery with specified controlled drugrelease rates, which can be adjusted through manipulating particle sizeand charge, and through amino alcohol bridging ester groups. This will(1) extend time between eye injections with improved clinical results inAMD, and (2) enable local treatment of OvCa and other cancers for moreeffective therapy.

Advantages

1. Anti-angiogenic adjuvant therapy delays relapse in ovarian cancer(OvCa) patients, but is based on agents whose anti-VEGF mechanism causegrave systemic toxicities. Our peptide anti-angiogenics are botheffective and safe because they mimic the action of endogenous, locallyexpressed anti-angiogenic protein (PEDF) whose levels dwindle in cancerand angiogenic eye disease.

2. OvCa patients receive chemotherapy by intravenous (IV) injections,but remission is more lasting with chemotherapy delivered directly theexisting anti-VEGF agents require IV administration, and are not locallyconcentrated or amenable to controlled release. Our peptideanti-angiogenics are specifically designed for slow localintraperitoneal release from carrier nano-devices.

3. Unexpectedly, in addition to eliminating neovasculature, our modifiedPEDF peptides kill OvCa cells but not normal ovarian cells.

4. Because our peptides use a mechanism distinct from VEGF inhibition,they can produce additive or synergistic results, and thus superiorclinical outcomes, in combination with existing anti-VEGFs.

5. Anti-VEGF proteins currently are used to treat wet maculardegeneration and other neovascular eye diseases by intraocularinjection. Their half-lives in the eye are between 4-6 days, they mustbe re-injected at 1-2 months intervals. This number of intraocularinjections is burdensome, painful, and poses risks of retinaldetachment, conical damage and infection. Our slow-release peptideformulations use linkage to transparent nanoparticles, with significantadherence to vitreous humor until the majority of the drug is released.This will give sustained action so that injections can be less frequent,ideally once every 2-4 months.

6. In our technology, intraperitoneal (IP) drug exposure is prolonged byuse of larger particles and/or by the adherence of the conjugates insmaller particles to poly-anionic mucosa/ECM or vitreous humor viasurface display of multiple positively charged peptides, in those casesof peptides having net positive charge (SEQ ID NO:4). This charge-basedanchoring allows prolonged drug release. Our delivery system offersmodes of local body space confinement using both size and charge, thusit is broadly applicable to cancer, endometriosis and eye disease. Incases where the vitreous gel of the eye has been surgically removed(vitrectomy) the slow delivery of carrier-linked peptides can be enabledthrough their formulation with pharmaceutical grade hyaluronic acid gelsfor intraocular use such as Healon® ophthalmic viscoelastics (OVDs). Inthe peritoneum our amino ester prodrug peptides can be linked as amidesto maleic anhydride activated carriers such as Gantrez™ polymersapproved for intraperitoneal administration.

Description

PEDF (Pigment Epithelial-Derived Factor) loss is a hallmark ofmalignancy/poor prognosis in human disease including advanced OvCa.PEDF, made in the retina, and in many tissues is a major inhibitor ofboth tumor and ocular angiogenesis and is reduced in eyes withneovascular disease. Importantly, added PEDF significantly impedes thecancer growth in preclinical models. PEDF's anti-angiogenic,pro-apoptotic activity was localized to its N-terminal 34 amino acidfragment. The anti-angiogenic, pro-apoptotic activity was furtherrefined to an 18 amino acid peptide (P18), (see U.S. Pat. No. 8,198,406)with pronounced anti-angiogenic and anti-cancer activity in pre-clinicalmodels; this short sequence comprises only 4% of the PEDF mass and wasrecently found to induce apoptosis in cultured OvCa cells. IntravitrealP18 also inhibits ocular neovascularization in preclinical AMD models.

We now have invented new technology which greatly improves on P18 bydiscovering much smaller and more practical active fragments within P18,with modifications allowing them to be linked to nano-device carriersthrough metastable ester bonds with a capacity to achieve a range ofrelease rates. The size and charge of the carriers allows them to beretained in body tissues, especially in the peritoneum and the eye forprolonged and focused action. The new peptides are made into prodrugsthrough ester bridging, directly, or through additional, more stablelinks to carriers. This involves the active amino acid sequences whereester forming di-carboxylic acid appendages retain or increaseanti-tumorigenic and anti-angiogenic activity. We also describe methodsfor esterifying and attaching these to carrier nanoparticles (see belowfor details). Also described are the methods of analysis to determinethe rates of ester breakdown and drug release, enabling choice of theoptimal ester form to give the desired half-life for a particulartherapeutic indication. Active amino acid sequences, which do not occurnaturally have led to unexpectedly more potent peptides with higher netpositive charge for prolonged retention in vitreous.

U.S. Pat. No. 8,198,406 disclosed the following sequence otherwisereferred to as “P18”:Acetyl-Asn-Phe-Gly-Tyr-Asp-Leu-Tyr-Arg-Val-Arg-Ser-Ser-Thr-Ser-Pro-Thr-Thr-Asn-amide(Ac-N-F-G-Y-D-L-Y-R-V-R-S-S-T-S-P-T-T-N-NH₂), with capped or uncappedtermini. A peptide this large is potentially immunogenic, expensive tomanufacture and difficult to isolate in high purity. It has too manyside chains to afford simple linkage chemistry as a prodrug. Thus itstherapeutic use would require daily injections at high cost. This couldbe overcome as a depot in ester prodrug form, thus coupled to alkylhydroxyl groups on carriers; furthermore, there are already 6 suchgroups potentially competing on the peptide itself. The one internal Aspcarboxyl group is not suited to ester formation, being unstable throughinternal peptide cyclization.

We have solved these problems by identifying the smaller activesub-region (underlined above) and by chemical modification, adding newcarboxyl groups and replacing the internal (Asp) carboxyl with a neutralamino acid. By testing anti-angiogenic activity in assays (blockingendothelial cell migration or induction of apoptosis), we comparedshorter fragments with full-size P18 and identified amino acids 3-11 asa fully active, potent core. We then examined the activity of the samepeptide appended with di-carboxylic acids as amides to the N-terminus tobe used as a practical linkage site, via succinic, glutaric and adipicacids, and also with beta-Alanine added to the C-terminus. We found itnecessary to add the N-methyl amino acid, Sarcosine (Sar) for couplingat the N-terminus to insure stability of succinic or glutaric amidehalf-esters that could cyclize rapidly when attached to ordinary(non-methylated) amino acids.

We discovered, by means of a caspase-3 activation assay, that theoptimal small peptides induce apoptosis in ovarian cancer (OvCa) cellsin vitro, and that this activity correlates with their potency inblocking the VEGF-stimulated sprouting of capillary tubes from smallsegments of explanted mouse choroid. This finding predicts activityagainst wet macular degeneration, retinopathy of prematurity anddiabetic retinopathy as well as activity against OvCa and possibly othercancers. Our optimal linkable carboxy-peptide, starting withglutaric-Sar, was unexpectedly found to be 3-5 fold more potent thanuncapped or succinyl peptide in these assays. Furthermore internalreplacement of negatively charged aspartate by neutral asparagine, whenglutarate or adipate is N-terminally appended, improved biologic potencyby up to 5-fold. Replacement of the Val-Arg-Ser sequence from within P18with Pro-ethylamide, Val-Pro-ethylamide or Pro-ethylamide, orVal-Gln-Ser also increased potency while lowering the peptide netcharge.

In order to obtain a range of ester prodrugs, we developed syntheses ofglutaryl or adipoyl Sar, Gly, or Sar-Gly modified peptides and theiresters with amino alcohol linkers, followed by stable attachment of theamino groups of the amino alcohol linkers as amides to carriers havingmultiple surface carboxyl groups. These include cross-linked hyaluronicacids, and carboxy dendrimers having 16, 32, or 64 carboxyl groups perparticle. The amino-alcohol esters can also link as amides to maleicanhydride-based polymers such as Gantrez™, which are approved forintraperitoneal use.

When carrier particles have uncharged surface groups as inpolysaccharides like dextran or in hydroxylated dendrimers, amino groupsof the amino alcohol linkers can be stably linked to surface hydroxylgroups as carbamates, The peptides can be released from the particlesvia ester hydrolysis of the ester bond between the carboxy-peptides andthe amino alcohol linkers.

Alternatively, for shorter ester hydrolysis half-lives (7-10 days) weinclude direct esterification of the adipoyl or glutaryl peptides tohydroxyl groups of poly-OH polymers like dextran, after the latter areactivated with mesitylene sulfonyl chloride. The peptides described hereare potently active 8-10 amino acid fragments of P18 with appendeddi-carboxyl groups where a free carboxyl group is directly esterified toeither (A) hydroxyl groups of polysaccharides (e.g. dextran) withparticle sizes ranging from 0.01 to 20 microns in diameter, or (B)bridging amino-alcohols where the amino groups are attached throughamide bonds to carboxyl groups of poly-carboxylated polymer carrierssuch as carboxylated dendrimers (G-1.5-G-4.5, 16-128 carboxyl groups perdendrimer) or nanogels derived from crosslinked hyaluronic acid or fromcondensed dextrans with diameters ranging from 0.01-20 microns. Animportant aspect of this construct is that peptide-loaded carriers(e.g., when loaded with SEQ ID NO:4, having a net charge of +2 whenesterified) have a positive zeta-potential, i.e. a net positive chargein order to immobilize them at their site of injection through multipleweak ionic interactions. This derives from the fact that each attachedpeptide, when esterified has a net charge of +1 or +2, and is especiallycritical for use in the eye, which contains high concentrations of theviscous poly-anionic hyaluronic acid (HA). Thus an ideal particle to beused for intravitreal injection will be less than 200 nm in diameter, asize that does not impede diffusion out of the eye when neutral ornegatively charged, but in our system will have net charge of +20 to+100 per particle, for long-term drug release. Ideally, >50% ofparticles injected in 0.05 ml should be contained at the injection siteby adherence to HA in the vitreous humor. The peptide-loaded carriermust be transparent for use in the eye, this is a property of nanogelnetworks and also of the much smaller dendrimers.

We identified active conjugable peptides, succinate-Sar-P3-11,glutarate-Sar-P3-11, adipate-Sar-P3-11 and adipate-P3-11 also shorterpeptides with only 8-9 amino acids, where the distal Arg of thesesequences is removed and replaced by neutral amino acids. All identifiedpeptides are at least as active as the parental P18 peptide.Importantly, these peptides are shorter than 12 amino acids, andtherefore not likely to be immunogenic. Their production cost and puritywill be practical, while the production and purification of largerpeptides is difficult and expensive. The amino acid sequence of SEQ IDNO:1 is identical to that found in human PEDF and in P18 and, like P18,has a net charge of +1. SEQ ID NO:1 has the advantage of being small.SEQ ID NOs: 2, 4, 6, 7, 8, 9 have the additional advantage of containingN-terminal carboxylates for ester linkage. SEQ ID NOs:4, 6, 7, 8, 9, 10,11, 12, 13 also have Asn (N) replacement of Asp (D), restoring theoriginal charge, which surprisingly enhances potency by 2-10 fold. SEQID NOs:7, 8, 9, 10, 12 also have replaced the second Arg with otherneutral amino acids, leading to a changed net charge of zero and 6-10fold increased potency. These new amino acid sequences are not known toexist in nature, containing 3-4 amino acids (Sar, Asn, Pro, Gln) notfound in this 9 amino acid segment of human PEDF or in any knownprotein.

For use in the eye, the ideal half-life of ester cleavage by simplehydrolysis at 37° C. and pH7.4 should be 10-100 days, 20-60 days beingideal. Approximately 1 mg of peptide attached to 0.1-1 mg of the carrierwould be delivered in the eye in 50 μl injection volume.

For simplified testing of release rate, amino alkyl or amino aralkylesters of various amino alcohols are made with adipoyl orglutaryl-Sar-Gly-amido rhodamine. These are attached to a carrierwith >10,000 MW overall molecular weight with total Absorbance at 550 nmmeasured after dialysis. Ester linkage to carrier may be direct or viaamino alcohol bridge. Examination of Absorbance at 550 nm released intofiltrate of spin filtration (10 kDa cutoff) at various days afterincubation gives the rate of ester breakdown in buffer. This is repeatedin the presence of animal vitreous humor or ascites fluid from mice withorthotopic ovarian cancer to confirm release rates. For IP delivery totreat OvCa or endometriosis 20-400 mg of carrier linked-peptide can bedelivered in 1-25 ml of injection fluid (buffer or saline).Sterilization will be most facile where loaded particles are <200 nmdiameter. Larger carriers may require gamma-ray sterilization. Ideal IPrelease rates for peptide should be in a similar range.

References

Kyoungmin Park, Ji Jin, Yang Hu, Kevin Zhou, Jian-xing Ma,“Overexpression of Pigment Epithelium-Derived Factor Inhibits RetinalInflammation and Neovascularization,” Am. J. Pathol 178(2): 688-698;2011.

Payam Zahedi, James Stewart, Raquel De Souza, Micheline Piquette-Miller,Christine Allen, “An injectable depot system for sustainedintraperitoneal chemotherapy of ovarian cancer results in favorable drugdistribution at the whole body, peritoneal and intratumoral levels,”Journal of Controlled Release, Volume 158, Issue 3, 28 Mar. 2012, Pages379-385.

Barber E L, Zsiros E, Lurain J R, Rademaker A, Schink J C, Neubauer N L,“The combination of intravenous bevacizumab and metronomic oralcyclophosphamide is an effective regimen for platinum-resistantrecurrent ovarian cancer,” J Gynecol Oncol. 2013 July; 24(3):258-64.

Filleur S, Volz K, Nelius T, Mirochnik Y, Huang H, Zaichuk T A, BecerraS P, Yap R, Veliceasa D, Shroff E H, Volpert O V, “Two functionalepitopes of pigment epithelial-derived factor block angiogenesis andinduce differentiation in prostate cancer,” Cancer Res. 2005;65:5144-52.

Mirochnik Y, Aurora A, Schulze-Hoepfner F T, Deabes A, Shifrin V,Beckmann R, Polsky C, Volpert O V, “Short pigment epithelial-derivedfactor-derived peptide inhibits angiogenesis and tumor growth,” ClinCancer Res. 2009; 15(5):1655-63.

Xu Q, Boylan N J, Suk J S, Wang Y Y, Nance E A, Yang J C, McDonnell P J,Cone R A, Duh E J, Hanes J, “Nanoparticle diffusion in, andmicrorheology of, the bovine vitreous ex vivo,” J Control Release. 2013Apr. 10; 167(1):76-84.

U.S. Pat. No. 8,530,416.

U.S. Pat. No. 8,278,284.

U.S. Pat. No. 8,198,406.

European Patent Publication No. 1265627.

Example 2—Small Peptides for the Treatment of Neovascular Disease,Inflammatory Disease and Cancer

We have discovered biologically active peptide sequences within PEDFincluding the 18-amino acids long P18. Novel modified peptides and newpeptide sequences disclosed herein are exemplified by SEQ ID NOs:1-13.SEQ ID NO:1 is an active truncation of P18 (see U.S. Pat. No. 8,198,406)having only 9 amino acids (i.e., ½ of amino acids in P18). FIG. 1provides ED₅₀ (50% effective dose) values for the peptides wherein ED₅₀is the measure of anti-angiogenic potency obtained by quantitativeassessment of apoptosis of activated human endothelial cells (EC) asdescribed in FIG. 3. SEQ ID NOs:2-11 are modifications of the nativePEDF sequence, which improve potency (ED₅₀) by 2-20 fold and enableformation of prodrugs. In preferred embodiments internal chargedresidues are removed or replaced by neutral amino acids, and N-terminalreplacement or addition of sarcosine is utilized to stabilize glutaricand adipic ester prodrugs. Replacement of anionic aspartate by neutralasparagine in SEQ ID NOs:4-13 leads to improved potency, which isfurther enhanced by N-terminal addition of dicarboxylic acids (glutaric,adipic, suberic), as illustrated in the more potent anti-tumor activityof SEQ ID NO:4, compared with SEQ ID NO:2 seen in FIG. 5A. Truncationsand specific amino acid changes at the C-terminus also enhance potency(ED₅₀), as exemplified in SEQ ID NO:10.

FIG. 2 shows that the described peptides reduce cell viability inmultiple human and mouse ovarian cancer cells, while normal ovarianepithelial cells (nOSE) and non-stimulated epithelial cells (EC) remainviable. As illustrated by the results of FIG. 2, ovarian cancer cells,normal ovarian epithelial cells (nOSE), and unstimulated mouseendothelial cells (mEC) were treated with increasing concentrations ofSEQ ID NO:2 and viability was measured using a WST assay. Thedifferences observed at 10 and 100 nM are statistically significantP<0.02, as determined by Wilcoxon log rank test (Graph Pad Prizmsoftware).

FIG. 3 illustrates the biological activity of various peptides atinducing apoptosis in endothelial cells at various concentrations. Themodified peptides of SEQ ID NOs:6, 8, 10, and 11 were compared to themodified peptide of SEQ ID NO:4 at the indicated concentrations.

FIG. 4 provides further experimental results illustrating the biologicalactivities of the disclosed peptides. In FIG. 4, the peptides'activities were measured using apoptosis of VEGF-stimulated endothelialcells as the measured property. The results confirm the selectivity ofpeptides' effects on both of activated (angiogenic) ECs (FIG. 4, Panel(B)) and on ovarian cancer cells (FIG. 4, Panel (D)) using apoptosis asmeasure of activity.

FIG. 5 shows the capacity of SEQ ID NO:2 and SEQ ID NO:4 to causeshrinkage in a mouse of human OVCAR3 tumors growing in the peritonealspace. Mice were implanted with 10⁶ ovarian cancer cells in the leftovary and tumors were allowed to reach progressive growth phase. Themice then were treated with continuous peptide administration (SEQ IDNO:4, 5 mg/kg) for 4 weeks. At the end of treatment, mice wereeuthanized, the tumors extracted and processed for analyses. Tumor masswas measured at the endpoint (Panel (A)). Representative tumors areshown in panel (B) and histopathology of tumors is presented in panel(C). Ovarian tumors from mice treated with SEQ ID NO:4 or controlvehicle were stained for the marker of blood vessels (CD31) orproliferation marker (Ki-67). We observed a visible decrease in thenumber of tumor blood vessels (FIG. 5, Panel (D)) and cell proliferation(FIG. 5, Panel (E)) in the presence of SEQ ID NO:4, compared to vehiclecontrol. In FIG. 5, panels (A-C) and B show reduced tumor mass andnormalized histology in the presence of SEQ ID NO:4; these effects areconsistent with the apoptotic effects of SEQ ID NO:4 on both activatedEC and OVCAR3 observed in vitro.

FIG. 6 shows the immunostimulatory effects of the SEQ ID NO:4 peptide inmacrophages which may be responsible for the anti-cancer activity of SEQID NO:4. In FIG. 6, Panels (A, B), normal ovarian epithelial cells (NOE)and ovarian cancer cells (Ovcar-3) were cultured alone or admixed withmacrophages (Raw264.7) at a 1:3 ratio. All cells were treated with SEQID NO:4 (10 nM) and cell death was visualized (Panel (A)) and quantified(Panel B)) using TUNEL stain. Panel (C) illustrates that tumor cellkilling was due to a macrophage-derived death molecule called TRAILwhich was induced by SEQ ID NO:4. This was demonstrated by adding TRAILinhibitor (DcR-1, decoy receptor-1) to the co-culture of ovarian cancercells and macrophages and measuring cell death (Apoptosis) as in Panel(A). In ovarian cancer, macrophages are a rich source ofimmunosuppressive molecule PD-L1, which interferes with ovarian cancercells recognition by T lymphocytes. Panel (D) illustrates that SEQ IDNO:4 attenuates immunosuppressive properties of tumor-associatedmacrophages by inhibiting the production of PD-L1.

FIG. 7 shows that SEQ ID NO:4 changes the subtype of macrophages to atumor-suppressive subtype and stimulates the infiltration of thetumor-suppressive macrophages into the ovarian tumors, which isassociated with increased ovarian cancer cell killing. Panel (A)illustrates decreased expression of mRNA for the marker oftumor-promoting macrophages (IL-10), and Panel (B) illustrates increasedmRNA levels of a marker for the tumor-suppressive phenotype (IL-12) inthe macrophages cultured in the presence of SEQ ID NO:4. Panel (C)illustrates that treatment with SEQ ID NO:4 eliminates thetumor-promoting macrophages (top, control treatment), while tumorsuppressive macrophages are prominent (bottom, treatment with SEQ IDNO:4). Panel (D) illustrates that treatment with SEQ ID NO:4 alsoincreases macrophage infiltration into tumors. Panel (E) illustratesthat, in tumors treated with SEQ ID NO:4, macrophages acquire theability to kill tumor cells in their proximity.

FIG. 8 shows the efficacy of described peptides in decreasing viabilityof canine osteosarcoma cells, by WST assay. Thus in addition to killingovarian cancer cells in vitro, as described in FIG. 2, concentrations ofthe tested peptides between 10 nM and 100 nM, also led to killing ofosteosarcoma cells. This shows broad anti-cancer activity of thepeptides, potency consistent with that of their causing apoptosis of EC.As with the latter, SEQ ID NO:10 and SEQ ID NO:11 show higher potencythan SEQ ID NO:4 and SEQ ID NO:8 in lowering the viability ofosteosarcoma cells. These results suggest that the disclosed peptidesmay be utilized in veterinary applications, for example, in treatingcanine cancer.

FIG. 9 shows efficacy of SEQ ID NO:4, SEQ ID NO:10, and SEQ ID NO:11peptides in mitigating laser induced CNV, an accepted mouse model ofexudative (“wet”) macular degeneration. Anesthetized mouse eyes wereinjected intravitreally with 1 μl PBS vehicle control or with 1 μl ofpeptide solution (4 mg/ml). After 2 days the same mice werere-anesthetized and 3 laser burns introduced onto the retina in each eyeto induce choroidal neovascularization (CNV). At 14 days post CNVinduction, sacrifice CNV areas were measured on choroidal-scleral flatmounts stained with fluorescent antibody against PECAM-1. The results inFIG. 8 illustrate that the peptides protect against neovascular retinaldamage for two weeks when 1 μl of 2-4 mM peptide is injected into mousevitreous two days prior to laser induction. This result implies theusefulness of the peptides in the treatment of neovascular eye disease.

FIG. 10 shows that for peptide SEQ. ID NO 10, having only 8 amino acidsand zero net charge at neutral pH, mitigation of laser-induced CNV canbe achieved by its administration as eye drops as well as by itsinjection into the vitreous. Twice daily eye drops (5 μl, 10 mM peptidein saline) begun 7 days before laser induction, then continued over 10of the ensuing 14 days, significantly reduced CNV compared to controlsreceiving saline eye drops. This demonstrated efficacy against ocularneovascular disease via eye drop administration.

FIG. 11 shows that in normal cells peptides show protective activityagainst cellular stressors. Panel (A) illustrates that the peptidesprotect renal cells (podocytes) from toxic effects of hyperglycemicstress. High glucose (20 mM and normal (5 mM) glucose conditions areshown for comparison. Panel (B) illustrates that the peptides protectretinal pigment epithelial cells (ARPE-1) against peroxide-inducedtoxicity. In particular, Panel (A) shows that SEQ ID NO:2 and SEQ IDNO:4 make retinal pigment epithelial (RPE) cells less susceptible tohydrogen peroxide, a reactive oxygen species (ROS) produced duringinflammation, as seen in dry macular degeneration. In Patent (B), kidneycells (podocytes) in vitro are sensitive to toxicity by high glucoseconcentration, which is mediated by endogenous production of ROS (seeFIG. 11 below). SEQ ID NO:4, at 10 nM, fully protects the kidney cellsfrom death implying that SEQ ID NO:4 is useful in protecting kidneydamage in diabetes.

FIG. 12 examines production of toxic ROS in kidney podocytes in vitro,by their exposure to high glucose, and to the chemotherapeutic agent,cisplatin, for which podocyte damage is a dose-limiting toxicity in thekidney. FIG. 10 illustrates that ROS production was stimulated byhyperglycemia (HG) or cisplatin (CPT) as “stressors.” Reactive oxygenspecies were detected using peroxide-specific stain by fluorescence.FIG. 10 also illustrates that SEQ ID NO:4 dramatically alleviates ROSproduction induced by both stressors. This implies the usefulness of SEQID NO:4 in protective therapy against kidney damage both in diabetes,and during chemotherapy.

FIG. 13 demonstrates that pro-fibrotic signaling in fibroblasts (inducedby TGF-b1) is suppressed by SEQ ID NO:4. Human primary dermalfibroblasts were activated with TGF-β1 (10 ng/ml), and SEQ ID NO:4 (30nM) was added where indicated or PEDF (20 nM) was used as control. After24 h the cells were lysed and pro-fibrotic signaling was assessed bymeasuring phosphorylation of TGB-β1 target, SMAD2/3. These results havepotential implications for the treatment of fibrotic diseases, such asidiopathic pulmonary fibrosis or renal fibrosis due to diabetes or otherinjury.

FIG. 14 gives examples of bridging amino-alcohols which, in theiramino-blocked forms (eg: N-BOC or N-FMOC amido-alcohols) can bemono-esterified to dicarboxylic acids (glutaric acid, adipic acid),which are then amide linked to the N-termini of peptides shown in FIG.1, during their solid-state synthesis. After de-blocking, release fromresin, and HPLC peptide purification the amino groups are then used toform carbamate or amide linkages to carrier particles. As indicated, inorder to obtain a wide range of ester prodrugs, we developed synthesesof glutaryl or adipoyl Sar, Gly, or Sar-Gly modified peptides and theiresters with amino alcohol linkers, followed by stable attachment of theamino groups of the amino alcohol linkers as amides to carriers havingmultiple surface carboxyl groups. These include cross-linked hyaluronicacids, and carboxy dendrimers having 16, 32, or 64 carboxyl groups perparticle. The amino-alcohol esters can also link as amides to maleicanhydride-based polymers such as Gantrez™ which are approved forintraperitoneal use. FIG. 14 shows examples of amino alcohols (Z groups)utilized to bridge peptide esters to a carrier, and a blockedcarbamato-ester of adipoyl sarcosine for N-terminal capping of a peptideduring solid state synthesis. This capping can be applied to thebridging groups shown to prepare dicarboxy-peptide esters ready forlinkage to carriers. In FIG. 14, FMOC or t-BOC protected carbamates ofthe above amino alcohols are esterified to dicarboxylic acid amides ofglycine or sarcosine. These carbamates then are appended to theN-termini of growing resin-bound peptide chains on resin during solidstate synthesis. Removal of the proximal protecting groups then allowsnew carbamate or amide attachments to be made to carrier polymersdisplaying hydroxyl or carboxyl groups on their surface such as dextranor hyaluronic acid.

FIG. 15 shows non-bridged carbamate linkage of dicarboxy peptides tohydroxylated carbohydrates, as activated by trimethylbenzenesulfonylchloride (MsCl). This yields ester prodrugs of the peptides having shorthalf-lives, compared to bridged forms (FIG. 14). When carrier particleshave uncharged surface groups as in polysaccharides like dextran or inhydroxylated dendrimers, amino groups of the amino alcohol linkers canbe stably linked to surface hydroxyl groups as carbamates. The peptidescan be released from the particles via ester hydrolysis of the esterbond between the carboxy-peptides and the amino alcohol linkers. FIG. 15shows direct esterification of a carboxypeptide to activated hydroxylgroups of a polymer. Esters obtained by this method are expected to haveshort half-lives, while secondary amino-alcohol esters, which may bebridged to carriers as carbamates or amides, show half-lives of esterrelease ranging from weeks to months. In FIG. 15, mesyl or tosylchlorides are used to charge sugar monomer OH groups. Mesyl/Tosyl groupsare then displaced, enabling attachment of dicarboxypeptides.

Example 3—Peptide Synthesis

Peptides were synthesized in the Simpson Querrey Institute's PeptideSynthesis Core at Northwestern University. Peptide synthesis was carriedout using a CEM Liberty microwave-assisted peptide synthesizer viastandard 9-fluorenyl methoxycarbonyl (Fmoc) solid-phase peptidesynthesis on rink amide MBHA resin. Peptides were purified byreverse-phase HPLC on a Varian Prostar 210 HPLC using awater/acetonitrile (each containing 0.1% v/v trifluoroacetic acid)gradient. Eluting fractions containing the desired peptide wereconfirmed by mass spectrometry using an Agilent 6520 LCMS. Confirmedfractions were pooled and the acetonitrile was removed by rotaryevaporation before freezing and lyophilization. Purity of lyophilizedproducts was tested by analytical HPLC on an Agilent 1200 HPLC.

For peptides HOOC—(CH₂)₃—CO-Sar-GYNLYRVRS—CONH2 andHOOC—(CH₂)₃—CO-Sar-GYDLYRVRS—CONH₂ (where Sar is sarcosine), then-terminal pentanoic acid was introduced after removal from theautomated synthesizer (HN-Sar-GYNLYRVRS-resin or HN-Sar-GYDLYRVRS-resin)via a manual coupling of 5-(tert-Butoxy)-5-oxopentanoic acid (2 equiv.relative to peptide) using HATU (1.9 equiv.) and DIEA (4 equiv.), shakenfor 4 h in DMF.

For peptides HOOC—C(CH₃)₂—(CH₂)₂—CO-Sar-Peg4-Sar-GYNLYRVRS—CONH₂ andHOOC—C(CH₃)₂—(CH₂)₂—CO-Sar-Peg4-RRYR—CONH₂ (where Peg4 is15-amino-4,7,10,13-tetraoxapentadecanoic acid), the n-terminalHOOC—C(CH₃)₂—(CH₂)₂—CO-Sar-Peg4 was introduced after removal from theautomated synthesizer (HN-Sar-GYNLYRVRS-resin or H2N-RRYR-resin) via thefollowing manual additions.15-amino-N-(9-fluorenylmethoxycarbonyl)-4,7,10,13-tetraoxapentadecanoicacid (2 equiv. relative to peptide) was coupled using HATU (1.9 equiv.)and DIEA (4 equiv.), shaken for 3 h in DMF. After removal of the Fmocwith 30% (v/v) 4-methylpiperidine in DMF for 30 min, Fmoc-sarcosine-OH(4 equiv. relative to peptide) was added with HBTU (3.9 equiv.) and DIEA(8 equiv.) and shaken in DMF for 3 h. Removal of the Fmoc was againperformed with the above 4-methylpiperidine solution before adding2,2-dimethylglutaric anhydride (2 equiv. relative to peptide) and DIEA(8 equiv.), which was shaken for 3 h or overnight in DMF.

Example 4—Synthesis of Aminoalkoxy Esters of Glutaric Acid or AdipicAcid as Bridge Structures Synthesis of Methyl5-[(2-tert-butoxy-2-oxoethyl)(methyl)amino]-5-oxopentanoate (Cmpd. 1)

One equivalent of Sarcosine tert-butyl ester hydrochloride was dissolvedin dichloromethane (DCM) with two equivalents of triethylamine (TEA)under stirring at room temperature. Then one equivalent of glutaric acidchloride monomethyl ester is dissolved in DCM and this solution is addeddropwise into the first solution. After 30 minutes, and solvent removalunder vacuum, the crude product is purified by column chromatography(CC) on silica gel (Davisil), eluting with a mixture of DCM:methanol.

Product Cmpd. 1:

Transparent oil. ¹H NMR (500 MHz, Chloroform-d) δ 4.16 (s, 1H), 3.64 (s,2H), 3.12 (s, 2H), 2.90 (t, J=5.4 Hz, 1H), 2.35 (t, J=5.5 Hz, 1H), 1.96(p, J=5.5 Hz, 1H), 1.43 (s, 5H).

2. Synthesis of potassium5-[(2-tert-butoxy-2-oxoethyl)(methyl)amino]-5-oxopentanoate (Cmpd. 2)

One equivalent of cmpd. 1 is dissolved in a mixture of methanol:water1:1. Then, 1 equivalent of potassium hydroxide is added to the firstsolution. Ester hydrolysis is complete after stirring, at roomtemperature for 30 minutes. This solution is extracted withwater:chloroform. The aqueous phase is evaporated under vacuum to givethe potassium salt.

Product Cmpd. 2:

Yellowish oil. ¹H NMR (500 MHz, DMSO-d₆) δ 4.11 (s, 1H), 2.96 (s, 2H),2.32 (t, J=7.1 Hz, 1H), 2.27 (t, J=7.1 Hz, 1H), 2.02 (p, J=7.1 Hz, 1H),1.42 (s, 5H).

3. Synthesis of 4-{[(tert-butoxy)carbonyl]amino}butyl4-{[2-(tert-butoxy)-2-oxoethyl](methyl) carbamoyl}butanoate (Cmpd. 3)

One equivalent of cmpd. 1 is dissolved in toluene under stirring atazeotropic reflux, mixed with a 6% of trioctylphosphinedimethylcarbonate and a 3% of lanthanum(III) nitrate and 1.5 equivalentsof 4-(Boc-amino)-1-butanol. After 6 hours the reaction is complete,solvent removed under vacuum and product purified by silica gelchromatography with a mixture of n-hexane:chloroform:methanol.

Product Cmpd. 3:

Yellowish oil. ¹H NMR (500 MHz, Chloroform-d) δ 5.34 (br, s 1H),4.18-4.12 (m, 2H), 3.24 (t, J=7.5 Hz, 1H), 3.09 (s, 2H), 2.90 (t, J=5.4Hz, 1H), 2.35 (t, J=8.1 Hz, 1H), 1.96 (tt, J=8.1, 5.3 Hz, 1H), 1.82-1.72(m, 1H), 1.68 (tt, J=7.6, 5.5 Hz, 1H), 1.43 (d, J=4.9 Hz, 9H).

4. Synthesis of2-[5-(4-{[(tert-butoxy)carbonyl]amino}butoxy)-N-methyl-5-oxopentanamido]aceticacid (Cmpd. 4)

One equivalent of cmpd. 3 is dissolved in a mixture ofDCM:trifluoroacetic acid 95:5 v:v at room temperature. After two hoursBOC ester removal is complete, after which the solution is diluted witha large volume of toluene and rotary evaporated using a 40-50° waterbath.

Product Cmpd 4:

Yellowish oil. ¹H NMR (500 MHz, DMSO-d₆) δ 7.13 (s, 1H), 4.15-4.07 (m,2H), 3.09-3.01 (m, 3H), 2.39-2.28 (m, 2H), 2.19-2.09 (m, 1H), 1.65-1.49(m, 2H), 1.39 (s, 5H).

5. Synthesis of4-{[(tert-butoxy)carbonyl]amino}cyclohexyl4-{[2-(tert-butoxy)-2-oxoethyl](methyl)carbamoyl}butanoate(Cmpd. 5)

One equivalent of cmpd. 1 is dissolved in toluene, with stirring atazeotropic reflux, mixed with 10% of Pentafluorophenylammonium triflate(PFPAT) and 1.5 equivalents of trans-4-(Boc-amino)cyclohexanol. After 6days the reaction is complete. The mixture is dried under vacuum andpurify it by silica gel chromatography, eluting with a mixture ofn-hexane:chloroform:methanol.

Product Cmpd. 5:

Yellowish oil. ¹H NMR (500 MHz, Chloroform-d) δ 4.45 (s, 1H), 4.16 (s,2H), 3.06 (s, 3H), 2.90 (t, J=5.4 Hz, 2H), 2.35 (t, J=5.4 Hz, 2H),2.04-1.92 (m, 6H), 1.67-1.57 (m, 2H), 1.43 (d, J=4.9 Hz, 20H).

6. Synthesis of2-{5-[(4-{[(tert-butoxy)carbonyl]amino}cyclohexyl)oxy]-N-methyl-5-oxopentanamido}aceticacid (Cmpd. 6)

One equivalent of cmpd. 5 is dissolved in a mixture ofDCM:trifluoroacetic acid 95:5 v:v at room temperature. After two hoursSar-BOC ester removal is complete, the solution is diluted with 10volumes of toluene and dried by rotary evaporation with mild heating(<50° C.). This product is then provided for N-terminal capping of apeptide attached to resin for solid state peptide synthesis.

Product Cmpd. 6:

Yellowish oil. ¹H NMR (500 MHz, Chloroform-d) δ 4.50-4.38 (m, 1H), 4.14(s, 1H), 3.02 (s, 2H), 2.90 (t, J=5.4 Hz, 1H), 2.35 (t, J=8.1 Hz, 1H),2.08-1.91 (m, 3H), 1.64 (dq, J=11.7, 6.7, 6.1 Hz, 1H), 1.56-1.43 (m,1H), 1.44 (s, 5H).

7. Synthesis of tert-butyl4-[(4-{[2-(tert-butoxy)-2-oxoethyl](methyl)carbamoyl}butanoyl)oxy]piperidine-1-carboxylate(Cmpd. 7)

One equivalent of cmpd. 2 and 0.1 equivalents of 18-crown-6 (phasetransfer catalyst) are dissolved in a mixture of dimethylsulfoxide:toluene 2:8 v:v, with stirring at 40° C. After 30 minutes, 1.1equivalents of 1-Boc-4-bromopiperidine and 0.1 equivalents of4-Dimethylaminopyridine (DMAP) are added. After 18 hours the reaction isdried under vacuum.

Crude product is stirred with water:chloroform. After water removal anda second water wash the organic phase is concentrated under vacuum andpurified by silica gel chromatography, eluting with a mixture ofn-hexane:chloroform:methanol.

Product Cmpd. 7:

Yellowish oil ¹H NMR (500 MHz, Chloroform-d) δ 4.69 (m, 1H), 4.18-4.09(m, 2H), 3.23 (dt, J=12.5, 7.2 Hz, 1H), 3.16 (s, 2H), 2.90 (t, J=8.0 Hz,1H), 2.35 (t, J=5.4 Hz, 1H), 2.08-1.82 (m, 3H), 1.45 (d, J=20.0 Hz, 9H).

8. Synthesis of2-[5-({1-[(tert-butoxy)carbonyl]piperidin-4-yl}oxy)-N-methyl-5-oxopentanamido]aceticacid (Cmpd. 8)

One equivalent of cmpd. 7 is dissolved in a mixture ofDCM:trifluoroacetic acid 95:5 at room temperature. Sar-BOC ester removalis complete after 2 hours, after which the solution is diluted with alarge volume of toluene and dried by rotary evaporation with mildheating.

Product Cmpd. 8:

Yellowish oil. ¹H NMR (500 MHz, DMSO-d6) δ 4.76 (m, 1H), 4.09 (s, 1H),4.03 (dt, J=12.5, 7.0 Hz, 1H), 3.11 (dt, J=12.6, 7.1 Hz, 1H), 2.96 (s,2H), 2.39-2.28 (m, 2H), 2.19-2.09 (m, 1H), 1.92-1.81 (m, 1H), 1.58 (dq,J=14.1, 7.1 Hz, 1H), 1.42 (s, 5H).

9. Synthesis of 3-{[(tert-butoxy)carbonyl]amino}cyclopentyl4-{[2-(tert-butoxy)-2-oxoethyl](methyl) carbamoyl}butanoate (Cmpd. 9)

One equivalent of cmpd. 1 is dissolved in toluene, with stirring atazeotropic reflux, and mixed with 10 mol % of Pentafluorophenylammoniumtriflate (PFPAT) and 1.5 equivalents ofN-[(1R,3R)-3-hydroxycyclopentyl]-, 1,1-dimethylethyl ester. After 6 daysthe trans-esterification is complete. The mixture is dried under vacuum,dissolved in 3 ml chloroform and purified by elution through silica gelwith a mixture of n-hexane: chloroform: methanol.

Product Cmpd. 9:

Yellowish oil. ¹H NMR (500 MHz, Chloroform-d) δ 5.12 (d, J=12.3 Hz, 1H),4.96 (p, J=7.0 Hz, 1H), 4.56 (s, 1H), 3.81-3.70 (m, 2H), 3.16 (td,J=12.5, 3.2 Hz, 1H), 3.10 (s, 3H), 2.61 (td, J=12.4, 1.5 Hz, 1H), 2.34(qdd, J=12.3, 3.2, 1.6 Hz, 1H), 2.30-2.11 (m, 5H), 2.03 (dq, J=12.7, 7.0Hz, 1H), 1.78-1.52 (m, 3H), 1.43 (d, J=4.9 Hz, 18H).

10. Synthesis of2-{5-[(3-{[(tert-butoxy)carbonyl]amino}cyclopentyl)oxy]-N-methyl-5-oxopentanamido}aceticacid (Cmpd. 10)

One equivalent of cmpd. 9 is dissolved in a mixture ofDCM:trifluoroacetic acid 95:5 at room temperature, de-blocking theSar-BOC ester in two hours. The solution is diluted with a large volumeof toluene and evaporated under vacuum with mild heating.

Product Cmpd 10:

Yellowish oil ¹H NMR (500 MHz, Chloroform-d) δ 4.87 (p, J=6.9 Hz, 1H),4.70 (d, J=12.4 Hz, 1H), 4.24 (s, 1H), 4.06 (p, J=7.0 Hz, 1H), 3.98 (d,J=12.5 Hz, 1H), 3.01 (s, 3H), 2.95 (ddd, J=12.5, 4.7, 1.8 Hz, 1H),2.65-2.50 (m, 2H), 2.31 (td, J=12.5, 2.5 Hz, 1H), 2.24-2.12 (m, 2H),2.06-1.94 (m, 1H), 1.95-1.83 (m, 2H), 1.78 (dt, J=13.0, 7.1 Hz, 1H),1.60-1.45 (m, 2H), 1.44 (s, 9H).

11. Synthesis of methyl6-[(2-tert-butoxy-2-oxoethyl)amino]-6-oxohexanoate (Cmpd. 11)

One equivalent of Glycine tert-butyl ester hydrochloride is dissolved indichloromethane (DCM) with two equivalents of triethylamine understirring at room temperature. Then one equivalent of Adipic acidmonomethyl ester chloride is dissolved in DCM and this solution is addeddropwise on the first solution. After 30 minutes the reaction mixture isconcentrated by evaporation, dissolved in 2-4 ml CHCl3 and the productis purified by column chromatography (silica gel, Davisil) with amixture of DCM:methanol.

Product Cmpd. 11:

Yellowish oil. ¹H NMR (500 MHz, Chloroform-d) δ 6.73 (s, 1H), 4.17 (s,1H), 3.64 (s, 2H), 2.52 (t, J=8.0 Hz, 1H), 2.35 (t, J=8.1 Hz, 1H),1.77-1.67 (m, 1H), 1.61 (pd, J=7.9, 1.1 Hz, 1H), 1.43 (s, 5H).

12. Synthesis of Potassium6-[(2-tert-butoxy-2-oxoethyl)amino]-6-oxohexanoate (Cmpd. 12)

One equivalent of cmpd. 11 is dissolved in a mixture of methanol:water1:1. Then, 1 equivalent of potassium hydroxide is added to the firstsolution. Conditions: stirring, room temperature. After 30 minutesmethyl ester hydrolysis is complete.

Crude product is washed in a separatory funnel with water:chloroform,the aqueous phase is evaporated under vacuum.

Product Cmpd. 12:

Yellowish oil. ¹H NMR (500 MHz, DMSO-d₆) δ 8.49 (s, 1H), 4.17 (s, 2H),2.30-2.24 (m, 2H), 2.16-2.10 (m, 1H), 2.13 (s, 1H), 1.56-1.45 (m, 4H),1.42 (s, 9H).

13. Synthesis of Tert-Butyl3-[(5-{[2-(tert-butoxy)-2-oxoethyl]carbamoyl}pentanoyl)oxy]pyrrolidine-1-carboxylate(Cmpd. 13)

One equivalent of the Comp 12 and 0.1 equivalents of 18-crown-6 aredissolved in a mixture of dimethyl sulfoxide:toluene 2:8. And stirred at40° C. After 30 minutes, 1.1 equivalents of 1-Boc-3-bromopyrrolidine and0.1 equivalents of DMAP are added. After 18 hours the reaction isstopped and dried under vacuum, the residue dissolved in CHCl3. This iswashed in a separatory funnel with water:chloroform. The organic phaseis concentrated under vacuum and purified by silica gel chromatography,eluting with a mixture of n-hexane:chloroform:methanol.

Product Cmpd. 13:

Yellowish oil. ¹H NMR (500 MHz, Chloroform-d) δ 6.73 (s, 1H), 4.96 (p,J=7.1 Hz, 1H), 4.90 (d, J=12.3 Hz, 1H), 4.04 (dd, J=9.4, 7.1 Hz, 1H),3.94 (d, J=12.3 Hz, 1H), 3.80 (dt, J=9.5, 7.1 Hz, 1H), 3.44-3.35 (m,2H), 3.04-2.88 (m, 2H), 2.67 (td, J=12.4, 4.2 Hz, 1H), 2.23 (dq, J=13.8,7.0 Hz, 1H), 2.21-2.08 (m, 1H), 2.11-1.90 (m, 3H), 1.45 (d, J=20.0 Hz,18H).

14. Synthesis of2-[6-({1-[(tert-butoxy)carbonyl]pyrrolidin-3-yl}oxy)-6-oxohexanamido]aceticacid (Cmpd 14)

One equivalent of cmpd. 13 is dissolved in a mixture ofDCM:trifluoroacetic acid 95:5 v:v at room temperature for 2 h,completely removing the Gly-t-But ester. The solution is diluted with 10volumes of toluene and dried by rotary evaporation with mild heating.

Product Cmpd. 14

Yellowish oil. ¹H NMR (500 MHz, Chloroform-d) δ 6.73 (s, 1H), 4.96 (p,J=7.1 Hz, 1H), 4.17-4.09 (m, 2H), 4.05 (d, J=12.3 Hz, 1H), 3.76 (dt,J=9.5, 7.1 Hz, 1H), 3.56 (dt, J=9.3, 7.1 Hz, 1H), 3.38 (dd, J=9.4, 7.1Hz, 1H), 2.98-2.90 (m, 1H), 2.61-2.49 (m, 1H), 2.46-2.35 (m, 2H),2.37-2.29 (m, 1H), 2.14-2.05 (m, 1H), 1.97 (dq, J=14.1, 7.1 Hz, 1H),1.77-1.57 (m, 3H), 1.47 (s, 9H).

15. Synthesis of 9H-fluoren-9-ylmethyl N-(4-hydroxycyclohexyl)carbamate(Cmpd. 15)

One equivalent of trans-4-aminocyclohexanol hydrochloride is dissolvedin DCM with two equivalents of TEA, and stirred in an ice bath. Then oneequivalent of 9-Fluorenylmethyl chloroformate (Fmoc-Cl) is dissolved inDCM and this solution is added dropwise to the first solution, reactionat room temperature being complete in 24 h.

After adding 5 volumes of CHCl3 crude product is washed in a separatoryfunnel with water, organic phase is evaporated under vacuum. Crudeproduct re-dissolved in a small volume of CHCl3 was then purified bysilica gel chromatography, eluting with a mixture ofn-hexane:chloroform:methanol.

Product Cmpd. 15:

White powder. ¹H NMR (500 MHz, Chloroform-d) δ 7.81 (dd, J=7.4, 1.6 Hz,2H), 7.59-7.49 (m, 4H), 7.43 (td, J=7.5, 1.5 Hz, 2H), 4.70 (d, J=5.8 Hz,2H), 4.50 (s, 1H), 4.37 (t, J=5.8 Hz, 1H), 3.74-3.63 (m, 2H), 2.10-1.93(m, 4H), 1.73-1.59 (m, 4H), 1.54 (s, 1H).

16. Synthesis of 9H-fluoren-9-ylmethyl N-(4-bromocyclohexyl)carbamate(Cmpd. 16)

One equivalent of cmpd. 15 is dissolved in dry tetrahydrofuran (THF)with one equivalent of tetrabromomethane, and stirred at roomtemperature, when the compounds are dissolved 1.0 equivalent ofethylenebis(diphenylphosphine) is added reaction is complete in 3 hours,after which the solution is filtered and the crude product is purifiedby silica gel chromatography, eluting with a mixture ofn-hexane:chloroform:methanol.

Product Cmpd. 16:

White powder ¹H NMR (500 MHz, Chloroform-d) δ 7.80 (dd, J=7.4, 1.6 Hz,2H), 7.58-7.49 (m, 4H), 7.43 (td, J=7.4, 1.6 Hz, 2H), 4.73-4.65 (m, 3H),4.64 (q, J=7.0 Hz, 1H), 4.27 (t, J=5.8 Hz, 1H), 4.11 (p, J=7.0 Hz, 1H),2.22 (dq, J=12.2, 6.9 Hz, 2H), 2.04-1.87 (m, 4H), 1.60 (dq, J=12.8, 6.9Hz, 2H).

17. Synthesis of 4-[(9H-Fluoren-9-yl)methoxycarbonylamino]cyclohexyl5-[N-methyl(tert-butoxycarbonylmethyl)amino]-5-oxovalerate (Cmpd. 17)

One equivalent of the cmpd. 2 and 0.1 equivalents of 18-crown-6 aredissolved in a mixture of dimethyl sulfoxide:toluene 2:8 v:v, withstirring at 40° C. After 30 minutes, 1.1 equivalents of cmpd 16 areadded. After 18 hours the reaction is stopped and dried under vacuum.Crude product dissolved in CHCl3 is washed in a separatory funnel withwater:chloroform, the organic phase concentrated by evaporation, thenpurified by silica gel chromatography, developed by a mixture ofn-hexane:chloroform:methanol.

Product Cmpd. 17:

¹H NMR (500 MHz, Chloroform-d) δ 7.83-7.75 (m, 3H), 7.56-7.39 (m, 5H),4.70 (d, J=7.0 Hz, 2H), 4.64 (s, 1H), 4.40 (p, J=6.9 Hz, 1H), 4.28 (t,J=7.0 Hz, 1H), 4.16 (s, 2H), 3.72 (p, J=6.9 Hz, 1H), 3.12 (s, 3H), 2.90(t, J=5.4 Hz, 2H), 2.35 (t, J=8.2 Hz, 2H), 2.11-1.90 (m, 6H), 1.73-1.62(m, 2H), 1.65-1.51 (m, 2H), 1.43 (s, 9H).

18. Synthesis of[N-Methyl(4-{4-[(9H-fluoren-9-yl)methoxycarbonylamino]cyclohexyloxycarbonyl}butyryl)amino]aceticAcid (Cmpd. 18)

One equivalent of cmpd. 17 is dissolved in a mixture ofDCM:trifluoroacetic acid 95:5 at room temperature, after 2 hours t-butylester removal is complete. The solution is diluted with 10 volumes oftoluene and dried by rotary evaporation with gentle heating.

Product Cmpd. 18:

¹H NMR (500 MHz, DMSO-d₆) δ 7.79 (dd, J=7.5, 1.7 Hz, 2H), 7.64 (dd,J=7.4, 1.5 Hz, 2H), 7.54 (td, J=7.5, 1.6 Hz, 2H), 7.50-7.39 (m, 3H),4.70 (d, J=7.1 Hz, 2H), 4.38 (p, J=7.0 Hz, 1H), 4.20 (t, J=7.1 Hz, 1H),4.09 (s, 2H), 3.85 (p, J=7.0 Hz, 1H), 2.89 (s, 3H), 2.34 (dt, J=19.7,5.3 Hz, 4H), 2.14 (p, J=5.3 Hz, 2H), 2.02 (dq, J=12.7, 7.0 Hz, 2H),1.70-1.59 (m, 2H), 1.60-1.49 (m, 2H), 1.26 (dq, J=13.0, 7.1 Hz, 2H).

In solid state peptide synthesis compound 18 is linked to the growingpeptide chain (eg: side chain blocked G-Y-N-L-Y-R-V-R-S-resin) linked toamide resin by standard coupling procedures. FMOC is then removed by 15minute exposure to 30% 4-methylpiperidine in DMF, after which blockinggroups and peptide are released by TFA to give4-amino-cyclohexanol-esterified SEQ ID 4 for carbamate or amide couplingto carriers.

References

Funatomi, T., Wakasugi, K., Misaki, T., & Tanabe, Y. (2006),“Pentafluorophenylammonium triflate (PFPAT), an efficient, practical,and cost-effective catalyst for esterification, thioesterification,transesterification, and macrolactone formation,” Green Chemistry,8(12), 1022-1027.

Stott, P. E., Bradshaw, J. S., & Parish, W. W. (1980), “Modified crownether catalysts. 3. Structural parameters affecting phase transfercatalysis by crown ethers and a comparison of the effectiveness of crownethers to that of other phase transfer catalysts,” Journal of theAmerican Chemical Society, 102(14), 4810-4815.

Hatano, M., & Ishihara, K. (2013), “Lanthanum (III) catalysts for highlyefficient and chemoselective transesterification. ChemicalCommunications,” 49(20), 1983-1997.

Pollastri, M P., Sagal, J. F., & Chang, G. (2001). The conversion ofalcohols to halides using a filterable phosphine source. TetrahedronLetters, 42(13), 2459-2460.

Example 5—Ester Hydrolysis Testing

Compounds (BOC amido alkoxy esters) was dissolved in a solution ofethanol. This solution was diluted with different buffers with range ofpH (from pH 7.33 to 8.34) to a known concentration. The sterile-filteredsolutions were stored at 37° C. and were checked by high performanceliquid chromatography (HPLC) over time points and the half life of theamido-alkoxy amino acid esters was calculated. (See Table 2).

TABLE 2 Half-life of amido-alkoxy amino acid esters at 37° C. t_(1/2)(days) Ester pH: 7.33 pH: 7.44 pH: 7.66 BOC-but-O-glt-Sar 20 — 11BOC-etOet-O-glt-Sar 8.5 — — BOC-pyrrol-O-adp-Gly — — 30BOC-piper-O-adp-Gly — — 23 BOC-trans3cyclohex-O-adp-Gly — — 70BOC-trans3cyclohex-O-glt-Sar — 27 — BOC-3cypent-O-glt-Sar — 29.66 —BOC-2cypent-O-glt-Sar — 20.97 — BOC-et-N-val-secO-glt-Sar — 23 —

Example 6—Generation of Dextran Micro- and Nano-Gels

4 g of dextran (M.w. 70,000, unit M.w. 190) was dried by co-evaporationwith anhydrous pyridine in vacuo and activated by reaction with 93 mg1,1′-carbonyldiimidazole (CDI) in 100 mL of anhydrous dimethyl sulfoxide(DMSO) at 25° C. under stirring for 4 h. Cholesteryl-amine wassynthesized by modification of cholesteryl chloroformate with a 3-foldexcess of 2,2′-(ethylenedioxy) bis (ethylamine) and purified by columnchromatography on silicagel using a stepwise gradient of methanol indichloromethane. 342 mg of cholesteryl-amine dissolved in 10 mL of DMSOwas added to the activated dextran, and reaction mixture was stirred for24 h at 25° C. The product (CDEX) was purified by dialysis insemi-permeable membrane tubes (MWCO 12-14,000) against water at 4° C.under stirring overnight, sonicated for 15 min and, then, freeze-dried.Total yield was 86%.

TABLE 3 Particle size, Yield, Micro/nanogel nm PDI SD % Treatment CDEX55 (100%) 0.27 4 86 Sonication

Example—7 Activation of Micro-Gels and Nano-Gels

Microgel and nanogels particles can be modified before charging withpeptides or peptide intermediates using chemical activation of hydroxylgroups on dextran/dextrin with 1,1′-carbonyldiimidazole (A), withtoluene or mesitylene sulfonyl chlorides (B) in order to obtaincarriers, which are ready to conjugation with peptides carriers.

210 mg CDEX was dried by co-evaporation with anhydrous pyridine andmixed with 36 mg 1,1′-carbonyldiimidazole (CDI) in 10 mL anhydrous DMSO.Reaction mixture was stirred for 4 h at 40° C. The activated CDEX waspurified by dialysis in semi-permeable membrane tubes (MWCO 12-14,000)against water at 4° C. under stirring overnight and, then, lyophilizedTotal yield of the imidazole-activated CDEX was 69%. Proton NMR showedthat 58 imidazole groups was attached to the polymer molecule (0.7 mmolimidazole moieties per 1 g).

350 mg CDEX was dried by co-evaporation with anhydrous pyridine, and 110mg toluenesulfonyl chloride dissolved in 25 mL anhydrous DMSO along with5 mL pyridine was added at cooling in ice bath. The reaction mixture wasstirred at 25° C. overnight. Pyridine was removed in vacuo byco-evaporation with methanol. The product was purified by dialysis insemi-permeable membrane tubes (MWCO 12-14,000) against water at 4° C.under stirring overnight and recovered after freeze-drying with a yieldof 75%. NMR spectrum analysis showed that 43 tosyl groups was attachedto the polymer molecule (0.52 mmol tosyl moieties per 1 g).

35 mg microgel was dried by co-evaporation with anhydrous pyridine anddissolved in 10 mL anhydrous DMSO-pyridine, 1:1. The mixture was cooledin ice bath, then 12 mg mesitylenesulfonyl chloride was added and themixture was stirred at 25° C. overnight. Pyridine was removed in vacuoby co-evaporation with methanol. The product was recovered by dialysisin semi-permeable membrane tubes (MWCO 12-14 kDa) against water at 4° C.under stirring overnight and freeze-drying with total yield of 70%. NMRspectrum analysis showed that 18 molecules of mesityl groups wasattached to the polymer molecule (0.22 mmol mesityl moieties per 1 g).

Example 8—Conjugation of Amino-PEG-Peptides

Amino-PEG(12)-peptides containing 1, 3 or 4 arginine residues (AP1, M.w.1,200; AP3, M.w. 1,375, and AP4, M.w. 1,550) have been conjugated withCDI-activated CDEX nanogel and investigated in biological systems.Urethane bonds formed in this reaction are stable in most biologicalenvironments.

Imidazole-activated CDEX (20 mg) was dissolved in 0.5 mL water, and pHwas adjusted to 8 with sodium bicarbonate solution. 9.6 mg of a 3-Argpeptide AP3, amino-PEG(12)-CO-Arg-Arg-Ser-Arg-amide, was dissolved in0.2 mL DMF and mixed with the CDEX solution. Reaction was continuedovernight at 25° C. and, then, quenched with 5 μL ethanolamine overnightat 4° C. Carrier-peptide conjugates were purified by dialysis insemi-permeable membrane tubes (MWCO 12-14,000) against water at 4° C.under stirring overnight and, then, freeze-dried. The reaction wasrepeated similarly for 9.6 mg AP3, and 10.9 mg AP4.

TABLE 4 Carbamate linkage of amino-PEG peptide with 3 Arg residuesPeptide, Size, Z-potential, Yield, Sample % nm (SD) PDI mV % CDEX-AP3 31124 ± 1.4 0.167 4.58 58

Example 9—Conjugation of Carboxyl-PEG-Peptides

Carboxyl groups of peptides and their derivatives have been conjugatedwith tosylated CDEX nanogel and investigated in biological systems.Ester bonds formed in this reaction are biodegradable, and free peptidesare slowly released in most biological environments.

Tosylated CDEX nanogels (6 and 12 mg) were dissolved in two separatevials in 0.25 and 0.5 mL DMSO, respectively, and, then, mixed with asolution of 6 mg carboxyl group-containing peptide (P868)adipic-G-V-D-alloIle-S-Q-I-R-P-ethylamide in 0.1 mL DMF and allowed tostand at 25° C. under stirring overnight. The reaction mixture wasquenched with glycine (5 mg) for 2 h at 25° C. and the product waspurified by dialysis in semi-permeable membrane tubes (MWCO 12-14,000)against water at 4° C. under stirring overnight and, then, freeze-dried.Total yield: 75-80%.

TABLE 5 Peptide, Size, Z-potential, Yield, Sample % nm (SD) PDI mV %CDEX-P868 high 29 112 ± 4  0.143 0.253 80 CDEX-P868 low 18 121 ± 1.20.158 0 75

Example 10—Characterization of CDEX-Peptide Conjugates

Peptide content in carrier-peptide conjugates was measured using aPierce BCA Protein Assay based on the calibration curve obtained withthe corresponding free peptide. Peptide analysis was performed asfollows: 20 mg/mL stock solution of peptide in water was used to prepareserial ½ dilutions of standards. Then, BCA working reagent (WR) wasprepared by mixing 50 mL of BCA reagent A (50 mL) with 1 mL of BCAreagent B. 25 μL of each standard dilutions and a carrier-peptideconjugate sample (3-5 mg/mL) were placed into 96-well plate intriplicates, then 200 μL of WR was added to each well, and the plate wasmixed thoroughly in shaker for 30 sec. Plate was covered and incubate at37° C. for 30 min, cooled at 25° C., and the absorbance was measured at562 nm using a plate reader. Peptide content (%) in the samples wascalculated based on the calibration curve of free peptide.

Sample characteristics (particle size, polydispersity andzeta-potential) were measured by a dynamic light scattering method usingMalvern Zeta Sizer Nano-S90 instrument according to the manufacturerrecommendations. Briefly, hydrodynamic diameter (d_(h)) andpolydispersity index (PDI) of nanogel/microgels were obtained for 1mg/mL aqueous solutions at 25° C. in triplicates after sonication for 30min and centrifugation at 12,000 rpm. Zeta-potential of the samples wasmeasured for the same solutions in standard 1 cm-cuvettesusingzeta-potential option in the company's software. Average of fivemeasurements±SD was registered.

In the foregoing description, it will be readily apparent to one skilledin the art that varying substitutions and modifications may be made tothe invention disclosed herein without departing from the scope andspirit of the invention. The invention illustratively described hereinsuitably may be practiced in the absence of any element or elements,limitation or limitations which is not specifically disclosed herein.The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention that in theuse of such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, but it is recognizedthat various modifications are possible within the scope of theinvention. Thus, it should be understood that although the presentinvention has been illustrated by specific embodiments and optionalfeatures, modification and/or variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention.

Citations to a number of patent and non-patent references are madeherein. The cited references are incorporated by reference herein intheir entireties. In the event that there is an inconsistency between adefinition of a term in the specification as compared to a definition ofthe term in a cited reference, the term should be interpreted based onthe definition in the specification.

We claim:
 1. A modified peptide comprising an N-terminal carboxyl groupand a modified amino acid sequence as follows:Z-B-X-AA0-AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9-AA10-Y  (SEQ ID NO:14)wherein: AA0 is absent or present, and when AA0 is present AA0 isselected from the group consisting of sarcosyl, and Gly; AA1 is absentor present, and when AA1 is present AA1 is selected from the groupconsisting of sarcosyl and Gly; AA2 is Tyr; AA3 is Asp or Asn; AA4 isLeu, Ile, or Val; AA5 is Tyr or Phe; AA6 is Arg; AA7 is Val or Pro; AA8is present or absent, and when AA8 is present AA8 is selected from thegroup consisting of Arg, Pro, and Gln; AA9 is present or absent, andwhen AA9 is present AA9 is selected from the group consisting of Ser andAla; AA10 is absent or present and when AA10 is present AA10 is selectedfrom the group consisting of Ser, and Ser-Thr, X is absent or present,and when X is present X is selected from the group consisting of acetyl,butyryl, hexanoyl, methoxy-PEG_((n))CO, hydroxy-PEG_((n))CO,amino-PEG_((n))CO, and sarcosyl-amino-PEG_((n))CO, where n is 3-13, andX is in an amide bond to an amino terminus of AA0, AA1, or AA2; B isabsent or present, and when B is present B is a di-carboxylic acidcontaining from 4-8 carbon atoms, which may be straight-chain orbranched, and B is in a half-amide bond to an amino terminus of X, or Bis in a half-amide bond to an amino terminus of AA0 when X is absent, orB is in a half-amide bond to an amino terminus of AA1 when X and AA0 areabsent, or B is in a half-amide bond to an amino terminus of AA2 when X,AA0 and AA1 are absent; and B may end in a free carboxyl group, or thisotherwise free carboxyl group may be in an ester bond to a hydroxylgroup of Z; wherein if B is present X is absent, and wherein if X ispresent B is absent; Z is absent or present if B is present, Z is absentif X is present, and when Z is present Z is joined through an ester bondto a carbonyl group of B, and Z is selected from the group consistingof: primary or secondary hydroxyl groups of sugar monomers present on apolymeric carbohydrate carrier; hydroxyl groups of a hydroxyl terminaldendrimer; and primary or secondary hydroxyl groups of acyclic or cyclicamino alcohols having between 4 and 6 carbon atoms and a single primaryor secondary amine that is protected through an amide bond or acarbamate bond; and Y is an amide or a substituted amide selected fromthe group consisting of amide or ethylamide.
 2. The modified peptide ofclaim 1 comprising a modified amino acid sequence as follows:B-AA0-AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9-Y  (SEQ ID NO:14).
 3. Themodified peptide of claim 1, wherein B is adipic acid or glutaric acidin half amide bond to AA0, or B is in a half-amide bond to an aminoterminus of AA1 when AA0 is absent, or B is in a half-amide bond to anamino terminus of AA2 when AA0 and AA1 are absent.
 4. The modifiedpeptide of claim 1, wherein B is present selected from the groupconsisting of succinic acid, glutaric acid, adipic acid, and subericacid.
 5. The modified peptide of claim 1, wherein the modified peptidehas a modified amino acid sequence selected from:sarcosyl-AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9-Y (SEQ ID NO: 14); andglutaryl-sarcosyl-AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9-amide (SEQ IDNO:14).
 6. The modified peptide of claim 5, wherein the modified peptidehas a modified amino acid sequence of:glutaryl-sarcosyl-Gly-Tyr-AA3-Leu-Tyr-Arg-Val-AA8-AA9-amide (SEQ IDNO:14).
 7. The modified peptide of claim 6, wherein the modified peptidehas a modified amino acid sequence selected from:glutaryl-sarcosyl-Gly-Tyr-Asp-Leu-Tyr-Arg-Val-Arg-Ser-amide (SEQ IDNO:2); glutaryl-sarcosyl-Gly-Tyr-Asn-Leu-Tyr-Arg-Val-Arg-Ser-amide (SEQID NO:4); glutaryl-sarcosyl-Gly-Tyr-Asn-Leu-Tyr-Arg-Val-Pro-ethylamide(SEQ ID NO:8).
 8. The modified peptide of claim 1, wherein the modifiedpeptide has a modified amino acid sequence selected from the groupconsisting of: adipoyl-sarcosyl-Tyr-Asn-Leu-Tyr-Arg-Val-Arg-Ser-amide(SEQ ID NO: 11); andadipoyl-sarcosyl-Tyr-Asn-Leu-Tyr-Arg-Val-Pro-ethylamide (SEQ ID NO:10).9. The modified peptide of claim 1, wherein the sequenceAA2-AA3-AA4-AA5-AA6 of SEQ ID NO: 14 is Tyr-Asn-Leu-Tyr-Arg (residues1-5 of SEQ ID NOs: 9-11).
 10. A prodrug comprising: (a) a carrierparticle comprising carboxyl groups or hydroxyl groups; and (b) themodified peptide of claim 1 conjugated to the polymeric carrierparticle.
 11. A prodrug of claim 10 in which the prodrug furthercomprises an amino alcohol linker that links the peptide to the carrierparticle, the peptide is esterified to a free hydroxyl group of thelinker, the linker is attached to the carrier particle via the aminogroup of the linker and a free hydroxyl group of the carrier particleforming a carbamate bond, or via the amino group of the linker and afree carboxyl group of the carrier particle forming an amide bond. 12.The prodrug of claim 11, wherein the linker is selected from the groupconsisting of amino-n-butoxy, amino-ethoxyethyloxy, amino-piperidyl(3,or 4)-oxy, amino-pyrrolidinyl(3)-oxy, amino-benzyloxy,BOC-aminoethylamido-valeric acid (4)-oxy, amino-cyclohexyl (3, or4)-oxy, and amino-cyclopentyl (3)-oxy.
 13. The prodrug of claim 10,wherein the carrier particle comprises dextrin, dextran, or hyaluronicacid.
 14. A pharmaceutical composition comprising the modified peptideof claim 1 or a prodrug thereof and a pharmaceutical carrier.
 15. Amethod of treating and/or preventing cancer in a subject in needthereof, the method comprising administering the pharmaceuticalcomposition of claim 14 to the subject.
 16. A method of treating and/orpreventing infectious disease in a subject having immune systemexhaustion characterized by elevated PD-L1, the method comprisingadministering the composition of claim 14 to the subject.
 17. A methodof treating and/or preventing an eye disease or disorder in a subject inneed thereof, the method comprising administering the composition ofclaim 14 to the subject.
 18. A method of treating and/or preventing akidney disease or disorder in a subject in need thereof, the methodcomprising administering the pharmaceutical composition of claim 14 tothe subject.
 19. A method of treating and/or preventing an ear diseaseor disorder in a subject in need thereof, the method comprisingadministering the pharmaceutical composition of claim 14 to the subject.